KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 466 KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 466

ULLA NUUTINEN

Mechanisms and Regulation of Apoptotic Pathways in Human Follicular Lymphoma Cells Aiming for Curative Treatment

Doctoral dissertation To be presented by permission of the Faculty of Medicine of the University of Kuopio for public examination in Auditorium ML3, Medistudia building, University of Kuopio, on Friday 11 th December 2009, at 12 noon

Institute of Clinical Medicine Department of Clinical Microbiology University of Kuopio

JOKA KUOPIO 2009

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Series Editors:

Professor Raimo Sulkava, M.D., Ph.D. School of Public Health and Clinical Nutrition Professor Markku Tammi, M.D., Ph.D. Institute of Biomedicine, Department of Anatomy

Author´s address: Institute of Clinical Medicine Department of Clinical Microbiology University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND E-mail: [email protected] Supervisors: Professor Jukka Pelkonen, M.D., Ph.D. Institute of Clinical Medicine Department of Clinical Microbiology University of Kuopio

Docent Pekka Riikonen, M.D. Department of Pediatrics Kuopio University Hospital

Reviewers:

Docent Juha Klefström, Ph.D. Cancer Cell Circuitry Laboratory, Biomedicum Helsinki University of Helsinki



Adjunct Professor Kaisa Heiskanen, Ph.D. Orion Corporation ORION PHARMA Turku

Opponent:

Professor Lea Sistonen, Ph.D. Turku Centre for Biotechnology, Department of Biology Åbo Akademi University

ISBN 978-951-27-1366-0 ISBN 978-951-27-1383-7 (PDF) ISSN 1235-0303 Kopijyvä Kuopio 2009 Finland

Nuutinen, Ulla. Mechanisms and Regulation of Apoptotic Pathways in Human Follicular Lymphoma Cells – Aiming for Curative Treatment. Kuopio University Publications D. Medical Sciences 466. 2009. 94 p. ISBN 978-951-27-1366-0 ISBN 978-951-27-1383-7 (PDF) ISSN 1235-0303 ABSTRACT Follicular lymphoma (FL) is still considered to be an incurable disease. The aim of this thesis was to study mechanisms and regulation of intrinsic and extrinsic apoptotic pathways induced by dexamethasone (Dex) and TRAIL, respectively, in human FL cell lines. In addition, the effect of an important FL microenvironmental signal, CD40 signaling, on TRAIL-, Dex- and doxorubicin-induced apoptosis was evaluated. Glucocorticoids (GCs) are commonly used in the treatment of lymphoid malignancies. However, the molecular mechanisms and regulation of GC-induced apoptosis remain still largely unknown. Mitochondria were necessary for Dex-induced apoptosis since overexpression of Bcl-XL prevented Dexinduced apoptotic changes. Interestingly, dominant negative (DN) caspase-9 also prevented Dex-induced apoptotic changes including the loss of mitochondrial membrane potential indicating that caspase-9 controls mitochondrial changes. New protein synthesis was required and the kinetics of the apoptotic events correlated with Dex-induced upregulation of the Bim. Interestingly, inhibition of glycogen synthase kinase-3 (GSK3) attenuated Dex-induced up-regulation of Bim and other apoptotic changes indicating that Dex-induced apoptosis is dependent on GSK3. Furthermore, inhibition of PI3-kinase-Akt pathway markedly enhanced and accelerated Dex-induced apoptosis already at the mitochondrial level by causing translocation of Bad to mitochondria. Thus, these results indicate that inhibitors of PI3kinase-Akt pathway might be combined with glucocorticoids to improve the treatment of FL. The extrinsic apoptotic pathway is activated through engagement of the cell surface death receptors by their ligands. TRAIL has emerged as the most promising for the death receptor targeted cancer therapy due to its remarkable feature of selectively inducing apoptosis in cancer cells without causing toxicity for normal cells. Based on Bcl-XL overexpression studies we identified type I (mitochondrial independent) and type II (mitochondria dependent) follicular lymphoma cell lines in response to TRAIL. Furthermore, a NF- B inhibitor, PDTC enabled TRAIL to activate type I apoptotic pathway in Bcl-XL overexpressing type II cells cells. However, an inhibitor of IKK did not switch apoptosis to type I pathway in type II cells, indicating that NF- B might not be responsible for the switch. The activation of apoptosis independently of mitochondria could have great advantage to treatment of FL because most cases of FL overexpress anti-apoptotic mitochondrial Bcl-2 protein. FL cells are malignant counterparts of germinal center (GC) B cells. Microenvironment of FL B cells has an important role in the progression of FL and might also have an impact on the treatment of FL. CD40 is an important mediator of microenvironmental survival signals in GCs. Therefore, we studied responses of CD40 signaling on TRAIL-, Dex- and doxorubicin-induced apoptosis in three human FL cell lines. In two of the FL cell lines, CD40 protected cells from apoptosis and the protection was entirely dependent on the activation of NF- B. In one of the FL cell lines, CD40 induced apoptosis itself. However, inhibition of NF- B itself induced apoptosis in all three FL cell lines. Therefore, our results indicate that inhibitors of NF- B or critical downstream anti-apoptotic targets of NF- or in some cases blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should be considered in the treatment of FL in order to prevent the protective effect of the microenvironment. National Library of Medicine Classification: QU 375, QZ 267, WH 200, WH 525 Medical Subject Headings: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Line, Tumor; Dexamethasone; Human; Lymphoma, Follicular; Mitochondria; NF-kappa B; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; 1-Phosphatidylinositol 3-Kinase

To my family

ACKNOWLEDGEMENTS

This work was carried out at the Department of Clinical Microbiology, Institute of Clinical Medicine, University of Kuopio, during the years 2003-2009. I wish to express my deepest gratitude to my principal supervisor, Professor Jukka Pelkonen, M.D., Ph.D., for his guidance, patience, open mind for new ideas and encouragement, during these years. I am deeply grateful to Jukka for opening the doors of the fascinating world of science to me and being there when I needed help and support. I am also deeply grateful to my second supervisor Docent Pekka Riikonen, M.D., for affording the clinical view for this research project. I wish to thank Docent Tuomas Virtanen, M.D., Ph.D., and Professor Jorma Ilonen M.D., Ph.D., for their scientific support and interesting discussions. I express my sincere gratitude to all my collaborators. I wish to thank Mine Eray M.D., Ph.D., for priceless collaboration, especially for the establishment of the human follicular lymphoma cell lines used in this study. I am also very grateful to Docent Jarmo Wahlfors, Ph.D., Riikka Pellinen, Ph.D., Tanja Hakkarainen, Ph.D., and Katja Häkkinen, M.Sc., for their fruitful collaboration and help with viral transductions. I owe my warmest thanks to all the present and former colleagues in the Department of Clinical Microbiology for their most valuable companionship, friendship and all their help during the years. Especially, my warmest thanks belong to Antti Ropponen, M.Sc., for his invaluable work contribution and help during this study. I am also deeply grateful to Mikko Mättö, Ph.D., for his guidance especially at the beginning of my research projects and help with flow cytometric methods. My special thanks belong to Jonna Eeva, M.D., Ph.D., for all the fascinating discussions, critical review of the manuscripts and her support and friendship. I also wish to thank all the other members of our research group Ville Postila, M.D., Anna-Riikka Pietilä, M.Sc., Maija Seppä, M.D., Sanna Suoranta, Niina Simelius, M.Sc., Vi Lieu and Jemal Adem, M.Sc., for their collaboration and help during this study. I am also deeply grateful to Pia Keinänen, Riitta Korhonen, Eila Pelkonen and Päivi Kivistö, for their excellent technical assistance and friendship. I am deeply grateful to the official reviewers of this thesis, Docent Juha Klefström, Ph.D., and Adjunct Professor Kaisa Heiskanen, Ph.D., for their time, careful and critical evaluation of the thesis, most valuable comments and interesting research discussions on the phone. I express my warm gratitude to all my dear friends for their support, discussions, laughs and cries, all the fun and enjoyable moments and adventures that we have experienced together. I also express my warmest thanks to all my relatives. I owe my sincere thanks to my parents, Kerttu and Pekka, for all the help, never-ending love, enormous support and encouragement during my whole life and just being there for me whenever I needed. I also wish to thank my grandfather Rudolf Böhme for all the support and love. I also want to express my sincere gratitude to my sister Eeva Kansala and her husband Kari Kansala for all the help, support and all the warm moments that we have shared, and my nieces Inka and Aada for bringing so much light, happiness and joy into my life. My warm thanks also belong to my sister Tiina Nuutinen for her support and all the joyful moments that we have shared.

This study was financially supported by the Kuopio University Hospital (EVO-fund), the University of Kuopio, North-Savo Cancer Organization and Paavo Koistinen Foundation, which are gratefully acknowledged.

Kuopio, December 2009

Ulla Nuutinen

ABBREVIATIONS

AIF ALL Apaf-1 Bcl-2 Bcl-XL BCR BH BIR CARD Caspase CHX c-IAP1 c-IAP2 CREB cypD Cyt c DD DED Dex DIABLO DISC DN DR GFP EMSA ERK FACS FADD FDC FL FLICE FLIP FOXO GC GR GRE GSK-3 HRP IAP IBM Ig I B IKK ILP-2 IRES JAK3

apoptosis inducing factor acute lymphoblastic leukemia apoptotic protease activating factor-1 B cell lymphoma 2 protein Bcl-2 related gene (large variant) B cell receptor BCL-2 homology baculoviral IAP repeat caspase recruitment domain cysteinyl aspartate-specific proteinase cycloheximide cellular IAP 1 cellular IAP 2 cyclic AMP-response element binding cyclophilin D cytochrome c death domain death effector domain dexamethasone direct IAP binding protein death inducing signaling complex dominant negative death receptor green fluorescent protein electrophoretic mobility shift assay extracellular signal regulated kinase fluorescence activated cell sorter Fas-associated death domain follicular dendritic cell follicular lymphoma Fas-associated death-domain like interleukin-1 converting enzyme FLICE inhibitory protein forkhead box O germinal center, glucocorticoid glucocorticoid receptor glucocorticoid response element glycogen synthase kinase-3 horseradish peroxidase inhibitor of apoptosis protein IAP-binding motif immunoglobulin inhibitor of B I B kinase IAP-like protein 2 internal ribosomal entry site Janus family kinase 3

JNK IMM LiCl mAb MAPK MHC ML-IAP MOMP NAIP NEMO NF- B NIK NLS NTD OMM OPG PBS PDTC PH PI3K PKB PLC PTEN PTP RING RIP RT-PCR SDS-PAGE SLO Smac STAT3 tBid TMRM TNF TNFR TRADD TRAF TRAIL t.u. VDAC XIAP m

JUN N-terminal kinase inner mitochondrial membrane lithium chloride monoclonal antibody mitogen activated protein kinase major histocombatibility complex melanoma IAP mitochondrial outer membrane permeabilization neuronal apoptosis inhibitory protein NF- B essential modulator nuclear factor B NF- B inducing kinase nuclear localization sequence N-terminal domain outer mitochondrial membrane osteoprotegerin phosphate buffered saline pyrrolidinedithiocarbamate pleckstrin homology phosphatidylinositol 3-kinase protein kinase B phospholipase C phosphatase and tensin homologue permeability transition pore really interesting new gene receptor interacting protein reverse transcriptase polymerase chain reaction sodium dodecyl sulfate-polyacrylamide gel electrophoresis secondary lymphoid organ second mitochondria-derived activator of caspase signal transduced and activator of transcription 3 truncated Bid methyl ester of tetramethylrhodamine tumor necrosis factor tumor necrosis factor receptor TNF-receptor type 1-associated death domain protein TNF receptor associated factor TNF-related apoptosis inducing ligand transducing unit voltage dependent anion channel X-chromosome-linked IAP mitochondrial membrane potential

LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which will be referred to in the text by Roman numerals. I

Nuutinen U, Postila V, Mättö M, Eeva J, Ropponen A, Eray M, Riikonen P, Pelkonen J. (2006) Inhibition of PI3-kinase-Akt pathway enhances dexamethasone-induced apoptosis in a human follicular lymphoma cell line. Exp Cell Res 312:322-330.

II

Nuutinen U, Ropponen A, Suoranta S, Eeva J, Eray M, Pellinen R, Wahlfors J, Pelkonen J (2009) Dexamethasone-induced apoptosis and up-regulation of Bim is dependent on glycogen synthase kinase-3. Leuk Res. 33(12):1714-7.

III

Nuutinen U, Simelius N, Ropponen A, Eeva J, Mättö M, Eray M, Pellinen R, Wahlfors J, Pelkonen J (2009) PDTC enables type I signaling in type II follicular lymphoma cells. Leuk Res. 33(6):829-36.

IV

Nuutinen U, Ropponen A, Eeva J, Eray M, Pellinen R, Wahlfors J, Pelkonen J (2009) The effect of microenvironmental CD40 signals on TRAIL- and drug-induced apoptosis in follicular lymphoma cells. Scand J Immunol. In press.

The original publications (I-IV) have been reproduced with the permission of the publishers. In addition, some unpublished data is presented.

CONTENTS

1. INTRODUCTION ..................................................................................................................... 17 2. REVIEW OF THE LITERATURE ......................................................................................... 18 2.1 B-cell development in periphery ........................................................................................... 18 2.1.1 Germinal centers ........................................................................................................... 18 2.1.2 CD40 signaling in germinal centers .............................................................................. 20 2.2 Follicular lymphoma ............................................................................................................. 21 2.2.1 General .......................................................................................................................... 21 2.2.2 Pathophysiology ............................................................................................................ 21 2.2.3 Treatment of FL ............................................................................................................ 22 2.3 Apoptosis............................................................................................................................... 22 2.3.1 General .......................................................................................................................... 22 2.3.2 Caspases ........................................................................................................................ 23 2.3.2.1 Inhibitor of apoptosis proteins (IAPs) ................................................................... 23 2.3.3 Intrinsic apoptosis ......................................................................................................... 25 2.3.3.1 Mitochondrial outer membrane permeabilization.................................................. 25 2.3.3.2 Bcl-2 family proteins in the regulation of MOMP ................................................ 26 2.3.3.2.1 Members of the Bcl-2 family ......................................................................... 26 2.3.3.2.2 Activation of BH3-only proteins ................................................................... 27 2.3.3.2.3 Bax and Bak activation .................................................................................. 27 2.3.4 Extrinsic apoptosis ........................................................................................................ 29 2.3.4.1 TRAIL and its receptors ........................................................................................ 29 2.3.4.2 Physiological role of TRAIL ................................................................................. 30 2.3.4.3 TRAIL-induced apoptosis signaling ...................................................................... 30 2.3.4.4 TRAIL-induced non-apoptotic signaling ............................................................... 32 2.3.4.5 Mechanisms of TRAIL-sensitivity and resistance ................................................. 32 2.3.4.5.1 Decoy receptors ............................................................................................. 33 2.3.4.5.2 Post-translational regulation of TRAIL-receptors ......................................... 33 2.3.4.5.3 FLIPs ............................................................................................................. 33 2.3.4.5.4 Other mechanisms ......................................................................................... 34 2.3.4.6 Clinical trials of TRAIL ........................................................................................ 34 2.4 Glucocorticoid-induced apoptosis ........................................................................................ 34 2.4.1 General .......................................................................................................................... 34 2.4.2 Glucocorticoid receptor and function ............................................................................ 35 2.4.3 Regulation of transcription specific genes .................................................................... 36 2.4.4 Mechanisms of glucocorticoid-induced apoptosis ........................................................ 36 2.4.5 The role of lysosomes in GC-induced apoptosis ........................................................... 37

2.5 Regulation of apoptosis by PI3-kinase-Akt-pathway ............................................................. 37 2.5.1 General........................................................................................................................... 37 2.5.2 Activation of PI3-kinase-Akt-pathway .......................................................................... 38 2.5.3 Anti-apoptotic targets of Akt ......................................................................................... 38 2.5.4 Glycogen synthase kinase-3........................................................................................... 40 2.5.4.1 General ................................................................................................................... 40 2.5.4.2 Regulation of glycogen synthase kinase-3 activity ................................................ 40 2.5.4.3 GSK3 promotes intrinsic apoptosis ........................................................................ 40 2.5.4.4 GSK3 inhibits extrinsic apoptosis .......................................................................... 41 3. AIMS OF THE STUDY ............................................................................................................. 42 4. MATERIALS AND METHODS .............................................................................................. 43 4.1 Cell lines and culture conditions (I-IV) ................................................................................. 43 4.2 Cell treatments (I-IV)............................................................................................................. 43 4.3 3H-thymidine incorporation ................................................................................................... 44 4.4 Propidium iodide staining (I-IV) ........................................................................................... 44 4.5 Enzyme assay for caspase activity (I) .................................................................................... 44 4.6 Detection of changes in mitochondrial membrane potential ( m) ...................................... 45 4.6.1 ApoAlert Mitochondrial Membrane Sensor Kit (I)........................................................ 45 4.6.2 TMRM staining (II, III) ................................................................................................. 45 4.7 FACS analysis of receptors ................................................................................................... 45 4.8 Preparation of samples for Western blot analysis (I, III, IV) ................................................ 46 4.9 Preparation of mitochondrial and cytosolic fractions (I-III)................................................. 46 4.10 Western blot analysis (I-IV) ................................................................................................. 46 4.10.1 Antibodies (I-IV) ......................................................................................................... 47 4.11 RT-PCR (IV) ........................................................................................................................ 47 4.12 Preparation of nuclear extracts (IV) ................................................................................... 48 4.13 EMSA assay (IV).................................................................................................................. 48 4.14 Cloning (II-IV) ..................................................................................................................... 49 4.15 Site-directed mutagenesis, cloning and production of lentiviruses (II-IV) .......................... 49 4.16 Lentiviral transduction, FACS analysis and sorting of GFP-positive cells (II-IV).............. 50

5. RESULTS AND DISCUSSION ................................................................................................ 51 5.1 Dexamethasone induces cell cycle arrest and apoptosis in dose-dependent manner (unpublished data) ...................................................................................................................... 51 5.2 Mechanism of Dex-induced apoptosis (I, II) ......................................................................... 53 5.3 Regulation of dexamethasone-induced apoptosis by PI3-kinase-Akt pathway (I) ................ 55 5.4 The role of glycogen synthase kinase-3 (GSK3) in Dex-induced apoptosis (II).................... 58 5.5 Molecular mechanisms of TRAIL-induced apoptosis (III) .................................................... 59 5.5.1 Type I and type II TRAIL signaling (III) ...................................................................... 62 5.6 The effect of microenvironmental CD40 signaling on dexamethasone-, doxorubicin- and TRAIL-induced apoptosis (IV) .................................................................................................... 68 6. CONCLUSIONS ........................................................................................................................ 72 7. REFERENCES .......................................................................................................................... 75

APPENDIX: ORIGINAL PUBLICATIONS

17 1. INTRODUCTION Follicular lymphoma (FL) is one of the most common non-Hodgkin’s lymphoma worldwide. In Finland, about 150 new cases are diagnosed annually. In the majority of FLs, the anti-apoptotic mitochondrial Bcl-2 protein is overexpressed due to t(14;18) chromosomal translocation. Microenvironment of FL B cells has also an important role in the progression of FL and might also have an impact on the treatment. FL is a heterogeneous and presently still incurable malignancy, characterized by a variable clinical course associated with frequent relapses and increasing chemoresistance to conventional anti-cancer regimens. Therefore new approaches are urgently needed for FL treatment. The life and death of cells must be balanced to maintain tissue homeostasis. Cells have an intrinsic mechanism to kill themselves via a process called apoptosis or programmed cell death. Apoptosis is genetically regulated process that eliminates billions of unwanted or damaged cells every day in the human body. Cells can undergo apoptosis via two different pathways: the intrinsic (mitochondrial) or extrinsic (death receptor) pathway. Activity of either of the pathways eventually leads to the activation of proteolytic cascade. Apoptosis is also involved in the regulation of many pathological processes including cancer. One of the hallmarks of human cancers is the evasion of apoptosis promoting tumor formation and progression. The ability of tumor cells to evade engagement of apoptosis can also play a significant role in their resistance to conventional therapeutic regimens. Most approaches used in cancer therapy including chemotherapeutics kill cancer cells by inducing apoptosis via the intrinsic pathway. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as the most promising for the death receptor targeted cancer therapy due to its remarkable feature of selectively inducing apoptosis in cancer cells without causing toxicity for normal cells. Recombinant TRAIL and monoclonal antibodies against TRAIL receptors are now in phaseI/II clinical trials. Extensive research in apoptosis field continues to solve detailed molecular mechanisms and regulation of apoptotic pathways, and is necessary to develop novel therapeutic strategies either to kill cancer cells selectively or lower the apoptotic threshold/overcome the resistance of cancer cells to molecules that induce apoptosis.

18 2. REVIEW OF THE LITERATURE 2.1 B-cell development in periphery The early B-cell development takes place in the bone marrow. B cells which have gained functional non-autoreactive surface B cell receptor (BCR) pass selection process and leave the bone marrow. These mature naive IgM and IgD expressing B cells circulate through the blood and lymph nodes until they contact specific antigen in the T cell area in the secondary lymphoid organs (SLOs) including spleen, lymph nodes, Peyer’s patches and tonsils. Antigens are brought to this location from peripheral sites by macrophages and/or dendritic cells. After antigen contact B cells stop migrating and remain in T cell zone (MacLennan, 1994). B cells that recognize the antigens, internalize antigen via the BCR complex, process it to peptides and present the peptide on MHC class II molecule to antigen specific helper T cells. Antigen specific T cells form stable conjugates with the antigen presenting B cells and is followed by soluble and membrane bound helper signals. These signals induce the B cells to proliferate and most of them terminally differentiate into antibody-secreting plasma cells in specialized extrafollicular sites (Klein and Dalla-Favera, 2008). The minority of activated B cells migrates into the primary follicles and start dividing to form germinal centers (Klein and Dalla-Favera, 2008). CD40 ligation (CD40 on B cells and CD40 ligand (CD154) on T cells) induces germinal center pathway. The role of other molecular interactions and cytokines remains controversial (MacLennan, 1994). 2.1.1 Germinal centers Germinal centers are divided into mantle, dark and light zones. The mantle zone contains recirculating B cells. Dark zone contains rapidly dividing centroblasts which undergo a massive clonal expansion. During proliferation the somatic hypermutation is activated (MacLennan, 1994). Somatic hypermutation introduces random point mutations into variable regions of the rearranged heavy- and light-chain genes at very high rate, giving rise to mutant immunoglobulin molecules on the surface of the B cells. Only some of the mutations increase the affinity of Ig for the antigen. Hypermutation is necessery for the affinity maturation of antibody (Klein and Dalla-Favera, 2008). Centroblasts continually give rise to the nondividing centrocytes of the light zone. Besides centrocytes, the light zone also contains most of the follicular dendritic cells (FDC) and CD4+ T cells that are present in germinal centers. Class switch recombination is thought to occur in light

19 zones of GCs. It ensures that the same variable region (the same antigen specificity) can be expressed with different constant region isotypes. Isotype switching is directed by cytokines released by helper T cells. CD40-CD154 interaction is also important for class switching (Stavnezer, 2000).

Mature B cell

Plasma cell IgM/D Mantle zone

Antigen presenting dendritic cell T helper cells

Dark zone

Clonal expansion Hypermutation centroblasts Light zone FDC

Class switch Positive and negative selection

centrocytes apoptosis

Memory cell

Germinal center Plasma cell

Figure 1. B-cell development in periphery (see text for details).

In the light zone, centrocytes undergo selection process where new antigen receptors are tested. Centrocytes which do not recognize the antigens anymore die by apoptosis. Centrocytes with best affinity antigen receptors are selected by taking the antigen from follicular dendritic cells. Then they process antigen and present it to T cells. T cells that recognize the antigens deliver two types of stimuli: contact mediated stimuli and activating cytokines that induce the proliferation and differentiation of B cells. B cells which have successfully bound antigen and survived the selection leave the germinal center to either become memory B cells or high affinity antibody secreting plasma cells (Klein and Dalla-Favera, 2008).

20 2.1.2 CD40 signaling in germinal centers CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is expressed on all mature B cells including germinal center B cells. In vivo, T cells provide CD40 ligand for CD40 engagement on B cells (van Kooten and Banchereau, 2000). CD40L exists as a trimeric ligand and the binding induces CD40 receptor oligomerization. This oligomerization appears to be a critical initiating step for CD40 mediated signaling (Kehry, 1996). CD40-mediated signaling is needed for B cell proliferation, isotype switching, germinal center formation and memory B cell commitment in response to T cell dependent antigens. CD40 signaling also rescues the germinal center B cells as well as immature B cells from BCR-mediated apoptosis (Dallman et al., 2003; Kehry, 1996). Mutations in the gene encoding CD40 ligand causes an immunodefiency disease called X-linked hyper-IgM syndrome. It is characterized by normal or elevated IgM levels but no IgG, IgA or IgE production, and defects in germinal center and memory B-cell formation (DiSanto et al., 1993; Korthauer et al., 1993). A similar phenotype is seen in CD40 or CD40L knockout mice (Dallman et al., 2003). Upon ligand binding, the cytoplasmic tail of CD40 delivers signals to the cells by recruiting TNF receptor-associated factors (TRAFs) 1, 2, 3, 5 and 6. The TRAF proteins activate different signaling

pathways

including

NF- B,

mitogen

activated

protein

kinases

(MAPKs),

phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC ) pathway. In addition, the Janus family kinase-3 (JAK3), which is associated with the cytoplasmic tail of CD40, undergoes phosphorylation, resulting in the activation of the signal transducer and activator of transcription 3 (STAT3) (Elgueta et al., 2009). The best-characterized signaling pathways activated by CD40 are the classical and the alternative NF- B signaling pathways (Berberich et al., 1994; Coope et al., 2002; Homig-Holzel et al., 2008). The ubiquitously expressed NF- B family of transcription factors includes NF- B1 (p105/p50), NF- B2 (p100/p52), RelA (p65), RelB and c-Rel (Hayden and Ghosh, 2004). The activity of NFB is inhibited through the interaction with members of the I B family of proteins. Activation and nuclear translocation of NF- B requires phosphorylation and proteosomal degradation of I Bs. In classical pathway, I B is phosphorylated by the I B kinase (IKK) complex that contains two catalytic subunits called IKK and IKK and a regulatory subunit called NEMO (NF- B essential modulator). The degradation of I B complex induces the translocation of NF- B heterodimers p50/p65 and p50/c-Rel to the nucleus. In contrast, the alternative NF- B pathway is activated

21 when NF- B inducing kinase (NIK) activates IKK , leading to processing of NF- B2/p100 to generate p52, which associates with RelB and translocates to the nucleus (Elgueta et al., 2009). Both signaling pathways regulate the transcription of several different genes by binding to consensus B sites in promoters (Hayden and Ghosh, 2004). NF- B transcription factors may have both anti-apoptotic and pro-apoptotic effects depending on the cell type and the type of the inducer. The anti-apoptotic target genes of NF- B include inhibitors of apoptosis proteins (IAPs), FLICEinhibitory proteins (FLIPs) and Bcl-XL (Lee et al., 1999; Travert et al., 2008; Wang et al., 1998). Although CD40 is considered a survival factor for normal B cells, in lymphoma B cells both CD40induced proliferation/survival and induction of growth arrest/apoptosis have been reported (Dallman et al., 2003). CD40 ligation has been shown to decrease the proliferation or induce apoptosis in high grade B cell lymphomas (Funakoshi et al., 1994; Wang et al., 2008). In contrast, in low-grade B cell malignancies CD40 has been shown to promote survival (Ghia et al., 1998; Travert et al., 2008). 2.2 Follicular lymphoma 2.2.1 General Follicular lymphoma (FL) is the second most common non-Hodgkin’s lymphoma worldwide. It is not curable and the median survival of FL patients is 8 to 10 years (de Jong, 2005). Most patients are over 50 years of age at the time of diagnosis. FL is equally common in both genders. FL is subdivided into three grades depending on the relative number of centroblasts in the lymphoma samples. Grade 3 is further subdivided into 3a and 3b. Excluding the more aggressive Grade 3b disease, FL is considered to be an indolent disease (Bendandi, 2008). Many patients are relatively asymptomatic at the time of diagnosis and have usually only painless peripheral lymphadenopathy. The patients with more advanced disease have symptoms such as fever, night sweats and weight lose. 2.2.2 Pathophysiology Majority of B cell lymphomas, including follicular lymphoma (FL), originate from GC B cells. Most cases of follicular lymphoma have a t(14;18) translocation. The translocation juxtaposes the bcl-2 gene on chromosome 18 and the enhancer region of the immunoglobulin gene from chromosome 14. The enhancer region causes over-expression of bcl-2 gene. The Bcl-2 is an inner

22 mitochondrial membrane protein that blocks apoptosis. However, the Bcl-2 overexpression itself is not sufficient to initiate tumorigenesis and other genomic alterations are needed for a malignant transformation (de Jong, 2005). The fact that FL cells undergo spontaneous apoptosis when cultured in vitro indicates that the microenvironment is also important for the development and progression of FL in vivo (Alvaro et al., 2006; Dave et al., 2004; Glas et al., 2005). Like normal germinal center B cells, FL cells interact through either soluble or membrane-bound signals with various immune cells, such as follicular dendritic cells, T cells and macrophages within the tumor microenvironment. CD40-CD40 ligand-interaction in combination with various cytokines has been shown to prolong the in vitro survival of FL cells (Eray et al., 2003; Ghia et al., 1998). Gene expression studies have shown that the nature of the tumor microenvironment predicts the survival of patients with FL and may influence the response to treatments and risk of transformation (Dave et al., 2004). The immune system may either promote or constrain tumor cell development, depending on the relative distribution and activation status of various cell populations. 2.2.3 Treatment of FL Clinical stage, prognostic factors and histological grading are needed to choose the best treatment. Asymptomatic FL patients with slow-growing disease are usually not treated until symptoms appear (wait-and-watch period). FL patients with a stage I or II disease are usually treated with radiation. Patients with advanced-stage disease (stage III and IV) are treated with combination chemotherapy or immunochemotherapy. In aggressive advanced stage FL chemotherapy is extensively used. Initial treatment consist usually chlorambucil with or without prednisone. In combination therapy, CVP (combination of cyclophosphamide, vincristine and prednisone) and CHOP (combination of cyclophosphamide, doxorubicin, vincristine and prednisone) have been widely used. A great improvenment in FL treatment has been achieved with the use of the chimeric monoclonal anti-CD20 antibody (Rituximab). Autologous or allogeneic transplantation have been used in young patients who relapse or show evidende of transformation (Bendandi, 2008). 2.3 Apoptosis 2.3.1 General Programmed cell death or apoptosis is cell’s ability to kill itself in a controlled way. Apoptosis is genetically regulated process and involved in regulation of many physiological and pathological

23 processes. During development, structures that are no longer needed are removed by apoptosis. Throughout life, apoptosis eliminates cells that are useless or potentially dangerous such as aged, infected, injured or mutated cells. Deregulation of apoptosis can lead to cancer (Jin and El-Deiry, 2005). Apoptosis involves a series of biochemical events leading to typical morphological changes, including cell shrinkage, membrane blebbing, cell membrane disruption, cytoskeletal rearrangement, chromatin condensation, and DNA fragmentation. Apoptotic cells are usually rapidly taken up and degraded by macrophages or neighboring cells before their intracellular contents leak into the extracellular space, thereby avoiding an inflammatory response (Jin and ElDeiry, 2005). 2.3.2 Caspases Apoptosis is mediated by intracellular cysteine proteases called caspases which share the ability to cleave their substrates after aspartate residues. To date, 14 mammalian caspases have been identified of which caspases-2, -3, -6, -7, -8, -9, and -10 have been shown to be involved in apoptosis (Li and Yuan, 2008). Caspases are produced as inactive zymogens containing a prodomain, a p20 large subunit and a p10 small subunit. Most of the caspases are activated by proteolytic cleavage. Active caspase is a heterotetramer, containing two small and two large subunits (Li and Yuan, 2008). Caspases are divided into initiator caspases (caspase-2, -8, -9, -10) and effector caspases (caspase3, -6, -7). Initiator caspases have long prodomains that contain the protein-protein interaction motifs, DED (death effector domain) or CARD (caspase recruitment domain). DED and CARD are involved in interacting with the upstream adaptor proteins. The short prodomain containing effector caspases are typically cleaved and activated by upstream iniatiator caspases. Active effector caspases are responsible for downstream execution steps of apoptosis by cleaving multiple cellular substrates (Li and Yuan, 2008). To date over 400 caspase substrates are identified (Luthi and Martin, 2007). There are two apoptosis signaling pathways, death receptor (extrinsic) and mitochondrial (intrinsic) pathway of which activation leads to activation of caspases. 2.3.2.1 Inhibitor of apoptosis proteins (IAPs) Enzymatic activity of caspases can be directly regulated by inhibitor of apoptosis proteins (IAPs). The IAP family of proteins consists of eight human analogues, including cellular IAP1 (c-IAP1),

24 cellular IAP2 (c-IAP2), X-linked inhibitor of apoptosis (XIAP), survivin, Apollon (also known as Bruce), melanoma IAP (ML-IAP, also known as Livin) and IAP-like protein 2 (ILP-2) (Hunter et al., 2007).

Figure 2. Structure of human IAPs.

All IAP proteins contain one or more BIR (baculoviral IAP repeat) domains which are thought to be responsible for caspase inhibition. In addition to the BIR domain, all IAPs except survivin contain one or more other functional domains, for example, RING domain, which possesses E3 ubiquitin ligase activity, and the CARD domain (Nachmias et al., 2004). XIAP is the best-described IAP and possibly the most potent suppressor of apoptosis. The linker region that precedes the BIR2-domain binds to active site of caspase-3 or -7 and prevents substrate binding. The BIR2 domain interacts with the N-terminus of the caspase small subunit. The BIR3 domain of XIAP binds to the IAP-binding motif (IBM) on the N-terminus of the small subunit of caspase-9, which is exposed upon proteolytic processing of caspase-9. Distinct part of BIR3 domain heterodimerizes with an interface of caspase-9, which is required for homodimerization of caspase-9. XIAP can also inhibit apoptosis through the E3 ubiquitin ligase activity of its RING domain that mediates proteosomal degradation of proteins, including caspases and I B (Fulda, 2009). In contrast to XIAP, the anti-apoptotic activity of c-IAP1 and c-IAP2 is thought not to be primarily related to direct inhibition of caspases. Rather, they interfere with caspase activation by targeting caspases or Smac/DIABLO to proteosomal degradation through their RING domain or binding to Smac/DIABLO sequestering it from XIAP, thus facilitating XIAP-mediated inhibition of caspases.

25 In addition, c-IAP1 and c-IAP2 promote survival by inducing activation of NF- B (Samuel et al., 2006; Fulda, 2009). 2.3.3 Intrinsic apoptosis Intrinsic or mitochondrial apoptotic pathway is triggered by multiple stimuli including DNA damage or cytotoxic drugs. The key event of mitochondrial pathway is mitochondrial outer membrane permeabilization (MOMP) which results in the release of mitochondrial intermembrane proteins such as cytochrome c (cyt c), second mitochondria-derived activator of caspase/ direct IAP binding protein (Smac/DIABLO), HtrA2/Omi, apoptosis inducing factor (AIF) and endonuclease D (EndoG) into cytosol. When released into the cytosol, cyt c binds to adaptor protein Apaf-1 (apoptotic protease activating factor-1). In the presence of dATP/ATP, the Apaf-1/cyt c complex oligomerizes into a heptameric structure resembling a wheel whose core contains seven N-terminal CARDs. The formation of this complex allows interaction of pro-caspase-9 with Apaf-1 through the interaction of its own CARD with the CARD of Apaf-1, thus placing individual pro-caspase-9 molecules in close proximity with one another. In apoptosome caspase-9 becomes an active initiator caspase and it further activates the effector caspases (caspase-3 and -7) which execute the cell death program (Riedl and Salvesen, 2007; Li and Yuan, 2008). Released Smac/DIABLO and serine protease HtrA2/Omi promote caspase activation by counteracting IAP-mediated caspase inhibition. AIF and EndoG induce caspase-independent cell death. AIF translocates to nucleus and causes chromatin condensation and high molecular weight DNA fragmentation (Susin et al., 1999). EndoG is also translocated to nucleus and induce internucleosomal DNA fragmentation (Li et al., 2001). 2.3.3.1 Mitochondrial outer membrane permeabilization Mitochondria are cellular organelles involved in energy production and are essential for cellular life. Mitochondria also play an important role in cell death. Mitochondrial outer membrane permeabilization (MOMP) is considered to be the “point of no return” during apoptosis as it results in the release of numerous apoptotic proteins that normally reside in the space between the outer (OMM) and inner (IMM) mitochondrial membranes to the cytosol (Chipuk et al., 2006). MOMP is frequently associated with the collapse of mitochondrial membrane potential ( the stimuli, the loss of

m

m).

Depending on

can occur before, during or after MOMP (Green and Kroemer, 2004).

26 The mechanisms responsible for MOMP during apoptosis remain controversial. The most important mechanism of MOMP under apoptotic conditions involves Bcl-2 family proteins. During apoptosis, activated Bax and/or Bak form pores in the outer mitochondrial membrane (OMM) resulting in the release of intermembrane proteins, such as cyt c, to the cytosol (activation of Bax and Bak discussed below). Inner mitochondrial membrane (IMM) can also participate in MOMP induction. Inner membrane can cause MOMP through permeability transition pore (PTP) which is a complex composed of several different proteins including VDAC (voltage dependent anion channel) in the OMM, ANT (the adenine nucleotide translocator) in the IMM and cyclophilin D (cypD) in the matrix. It has been suggested that opening of this pore leads to an influx of ions into the mitochondrial matrix causing the loss of

m,

and swelling of the matrix as water enters leading to rupture of OMM and

MOMP (Chipuk et al., 2006; Green and Kroemer, 2004). However, PTP function in apoptosis is still unclear because of swelling of mitochondrial matrix is not always a feature of apoptotic cells and release of cyt c may precede or even occur in the absence of the loss of

m

(Skommer et al.,

2007). Instead, PTP formation appears to engage necrotic cell death (Green, 2005). 2.3.3.2 Bcl-2 family proteins in the regulation of MOMP 2.3.3.2.1 Members of the Bcl-2 family The Bcl-2 family of proteins is divided into three groups based on their function and the number of BCL-2 homology (BH) domains present. The anti-apoptotic members (Bcl-2, Bcl-XL, Bcl-w, Mcl1, A1 and Bcl-B) are associated with the outer mitochondrial membrane and protect cells against a variety of apoptotic stimuli. They contain up to four BH domains (BH1-BH4). The pro-apoptotic members are divided into two groups, Bax-like multidomain (BH1-BH3) apoptotic proteins (Bax, Bak, Bok) and BH3-only proteins (Bik, Bid, Bad, Puma, Noxa, Bim, Hrk, Bmf). It is generally believed that Bcl-2 family proteins form homo- and heterodimers and the interactions between proand anti-apoptotic members neutralize the activity of each other (Adams and Cory, 2001; Bouillet and Strasser, 2002; Korsmeyer, 1999; van Delft and Huang, 2006). Subsequently the ratio of proapoptotic to anti-apoptotic Bcl-2 family members is critical in determining whether the cell will undergo apoptosis.

27 2.3.3.2.2 Activation of BH3-only proteins Activity of BH3-only proteins is regulated by several mechanisms at the transcriptional or at the post-translational level. At least four BH3-only proteins are transcriptionally induced in response to apoptotic stimuli. These include Hrk (Imaizumi et al., 1997; Imaizumi et al., 1999; Inohara et al., 1997), Puma (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001), Noxa (Oda et al., 2000) and Bim (Dijkers et al., 2000; Harris and Johnson, 2001; Putcha et al., 2001; Shinjyo et al., 2001; Whitfield et al., 2001). Bad, Bim and Bik are regulated by phosphorylation (Zha et al., 1996; Leung et al., 2008; Rambal et al., 2009; Verma et al., 2001). Pro-apoptotic potency of Bad and Bim is decreased by phosphorylation. On the contrary, phosphorylation of Bik enhances its proapoptotic activity (Verma et al., 2001). Bid is activated by proteolytic cleavage (Li et al., 1998; Luo et al., 1998) and targeting of truncated Bid (tBid) to mitochondria is facilitated by N-myristoylation at a site that becomes available for modification after cleavage (Zha et al., 2000). Bmf and Bim are regulated by sequestration to cytoskeletal structures (Puthalakath et al., 1999; Puthalakath et al., 2001). Bmf is sequestered to the actin cytoskeleton-based myosin V motor complex through its interaction with dynein light chain DLC2 (Puthalakath et al., 2001). Bim has three isoforms of which BimL and Bim EL are sequesterd to the microtubular dynein motor complex through interaction with DLC1/LC8. BimS does not bind to DLC1 and this appears to be reason why it has greater killing potency than BimL and BimEL (Puthalakath et al., 1999). 2.3.3.2.3 Bax and Bak activation Bax and Bak are constitutively expressed and induce MOMP following apoptotic stimuli suggesting that they are inactive in non-apoptotic cells (Kuwana and Newmeyer, 2003; Letai et al., 2002). Bax proteins can be found as monomers in the cytosol or loosely associated with the outer mitochondrial membrane when not activated. Bax translocates to and stably inserts into the mitochondrial outer membrane during the activation process (Billen et al., 2008; Hsu et al., 1997; Wolter et al., 1997). Bak is inserted into the outer mitochondrial membrane even when not activated (Wei et al., 2000). Bax or Bak are necessary for mitochondrial apoptosis since cells from mice lacking both Bax and Bak are resistant to a wide range of pro-apoptotic insults (Wei et al., 2001; Zong et al., 2001). The BH3-only proteins are shown to be essential initiators of apoptosis and they act upstream of Bax and Bak (Cheng et al., 2001; Huang and Strasser, 2000; Zong et al., 2001). Activation of certain BH3-only proteins is required for Bax and Bak to oligomerize and insert stably into the OMM.

28 There are two models how BH3-only proteins activate Bax and Bak. The direct activation model suggests that certain BH3-only proteins, namely Bim, tBid and Puma, act as activators by binding directly to Bax and Bak and promoting their activation (Cartron et al., 2004; Kim et al., 2006; Kuwana et al., 2005; Letai et al., 2002; Marani et al., 2002; Oh et al., 2006; Walensky et al., 2006). In this model, the rest of the BH3-only proteins act as sensitizers and bind to the anti-apoptotic Bcl2 members freeing Bim, tBid and Puma to activate Bax and Bak. The indirect activation model suggests that all BH3-only proteins bind to anti-apoptotic Bcl-2 family proteins replacing Bax and Bak which leads to activation of Bax and Bak (Chen et al., 2005; Willis et al., 2005; Willis et al., 2007). In this model, Bim, tBid and Puma are more potent inducers of apoptosis since they can bind to all antiapoptotic Bcl-2 proteins (Certo et al., 2006; Chen et al., 2005; Kuwana et al., 2005; Willis et al., 2005).

Figure 3. Direct and indirect activation model of how BH3-only proteins activate Bax/Bak.

29 Several recent findings strongly challenge the direct activation model (Willis et al., 2007). Regardless which model of BH3-only function is correct, both lead to the activation of Bax and/or Bak. One of the steps involved in the activation of Bax and Bak is a conformational change that exposes the N-terminus of these proteins. Next step in this process is the formation of Bax and Bak homo-oligomers that participate in forming pores and cause MOMP. However, the mechanisms by which Bax and/or Bak form pores at the OMM and mediate MOMP are not fully understood. 2.3.4 Extrinsic apoptosis The extrinsic apoptotic pathway is activated through engagement of the cell surface death receptors by their ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) superfamily. The members of this family are type I transmembrane proteins and are characterized by similar cysteine-rich extracellular domains which defines their ligand binding. Death receptors contain an intracellular 80-amino-acid-long region called “death domain” (DD) which is essential for transduction of the apoptotic signal. The most studied death receptors are Fas (CD95/Apo-1), TNFR1, TRAIL-R1 (DR4) and TRAIL-R2 (DR5/Killer/TRICK2). The ligands (FasL, TNF and TRAIL) that bind to the death receptors are structurally related proteins that belong to the TNF superfamily. These death ligands are mainly expressed as type II transmembrane proteins. In some cases, these proteins can be proteolytically cleaved and released, although the apoptosis inducing capacity of soluble ligands is significantly lower than of membrane-bound ligands (Guicciardi and Gores, 2009). 2.3.4.1 TRAIL and its receptors TRAIL was originally identified through sequence homology to the extracellular domain of FasL and TNF (Pitti et al., 1996; Wiley et al., 1995). TRAIL forms homotrimers and interacts with three cross-linked receptor molecules on the surface of the target cells. The biologically active conformation of TRAIL is stabilized by a zinc ion positioned at the trimer interface (Hymowitz et al., 2000). TRAIL induces apoptosis upon binding to its death domain containing receptors, TRAIL-R1 or TRAIL-R2 (Chaudhary et al., 1997; Pan et al., 1997b; Walczak et al., 1997). TRAIL can bind also to three other receptors which are unable to induce apoptosis and act as decoys. Decoy receptors TRAIL-R3 (LIT, DcR1) and TRAIL-R4 (TRUNDD, DcR2) are expressed on the surface of the cells but neither of them have a functional intracellular death domain (Marsters et al., 1997; Pan et al., 1997a). The fifth TRAIL receptor is a soluble osteoprotegerin (OPG) (Emery et

30 al., 1998). However, TRAIL binding affinity for this receptor is low at physiological temperatures (Truneh et al., 2000).

Figure 4. TRAIL receptors (see text for details).

2.3.4.2 Physiological role of TRAIL The physiological function of TRAIL is largely unclear. TRAIL-R- and TRAIL-deficient knockout mice are viable and fertile with no obvious developmental defects. TRAIL-R deficient mice have enhanced innate immunity responses, indicating negative regulation of the innate immune system via TRAIL (Diehl et al., 2004). Furthermore, TRAIL contributes to host immunosurveillance against primary tumor development and metastasis (Cretney et al., 2002; Schmaltz et al., 2002; Seki et al., 2003; Takeda et al., 2001). TRAIL knockout mice are more susceptible to tumor initiation following carcinogen treatment and to tumor metastasis (Cretney et al., 2002) and demonstrate an accelerated development of hematological malignancies (Zerafa et al., 2005). TRAIL has also been implicated in the regulation of autoimmunity (Lub-de Hooge et al., 2005). 2.3.4.3 TRAIL-induced apoptosis signaling Binding of trimeric TRAIL or agonistic monoclonal antibodies to TRAIL-R1 or TRAILR2 results in receptor oligomerization on the cell membrane and initiation of apoptosis. The intracellular signal transduction cascades triggered by FasL and TRAIL follow similar pathways. Activation of

31 the receptors leads to formation of a complex of proteins called death-inducing signaling complex (DISC). DISC involves adaptor protein Fas associated death domain (FADD), which binds via its own DD to the DD of the receptor and procaspase-8, which is recruited by FADD via interaction of death effector domains (DEDs) that are present in both FADD and procaspase-8. At the DISC, procaspase-8 is activated by auto-processing (Bodmer et al., 2000; Kischkel et al., 1995; Kischkel et al., 2000; Sprick et al., 2000). In humans, caspase-10 can also be recruited to DISC and promote apoptosis (Kischkel et al., 2001). In Fas-induced apoptosis, the activation of caspase-8/10 may, depending on the cell type (type I or II), lead either to direct activation of the downstream effector caspases or to amplification of the signal by activation of the mitochondrial pathway (Scaffidi et al., 1998). In type I cells, strong caspase-8/(10) activation at the DISC activates downstream effector caspases, such as caspase-3,-6 and -7, independently of mitochondria. In type II cells, caspase-8 activation is less efficient and promotes cleavage of pro-apoptotic Bcl-2 family protein Bid. Truncated Bid (tBid) translocates to mitochondria causing the depolarization of mitochondrial membrane and subsequent release of mitochondrial apoptotic factors, such as cyt c to cytosol (Li et al., 1998; Scaffidi et al., 1998). Subsequently, the apoptosome is formed leading to activation of caspase-9. Activated caspase-9 triggers downstream effector caspase activation, finally leading to apoptosis (Srinivasula et al., 1998; Zou et al., 1999). It should be noted that in both type I and type II cells loss of mitochondrial membrane potential and release of apoptotic factors from mitochondria are activated. However, only in type II cells overexpression of Bcl-2 or Bcl-XL prevents apoptosis (Srinivasula et al., 1998; Zou et al., 1999). Therefore, in type II cells apoptosis is mitochondria dependent and in type I cells apoptosis can proceed independently of mitochondria. TRAIL-induced apoptosis has also been shown to use both mitochondrial dependent and independent pathways (Ozoren and El-Deiry, 2002; Suliman et al., 2001). Overexpression of Bcl-2 or Bcl-XL does not block TRAIL-induced apoptosis in some cancer cell lines (Fulda et al., 2002; Keogh et al., 2000; Kim et al., 2001; Rudner et al., 2001; Walczak et al., 2000). In contrast, overexpression of Bcl-2 or Bcl-XL blocks or reduces TRAIL-induced apoptosis in other cancer cell lines (Fulda et al., 2002; Hinz et al., 2000; Munshi et al., 2001; Rokhlin et al., 2001; Ruiz de Almodovar et al., 2001; Srinivasula et al., 2000; Sun et al., 2001).

32

Figure 5. Simplified model of type I and type II signaling pathways in TRAIL-induced apoptosis.

Interestingly, recently the lysosomal pathway and cathepsin B has also been connected to TRAILinduced apoptosis (Guicciardi et al., 2007; Nagaraj et al., 2006; Werneburg et al., 2007). 2.3.4.4 TRAIL-induced non-apoptotic signaling In addition to triggering a pro-apoptotic signaling, TRAIL can activate multiple signal transduction pathways such as MAPKs (extracellular signal regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38), PI3-kinase-Akt and NF- B. Various combinations of proteins are involved in activation of these pathways by TRAIL. These proteins include FADD, TNF-receptor type 1associated death domain protein (TRADD), c-FLIP, caspases 8 and 10, TRAF2, NEMO and receptor interacting protein (RIP) (Johnstone et al., 2008). 2.3.4.5 Mechanisms of TRAIL-sensitivity and resistance TRAIL has been shown to induce apoptosis effectively in cancer cells with little or no toxicity against normal cells. However, not all tumors are sensitive to TRAIL. Sensitivity of TRAILinduced apoptosis can be regulated at several levels.

33 2.3.4.5.1 Decoy receptors Decoy receptors TRAIL-R3 and TRAIL-R4 can bind TRAIL but lack intracellular death domain and therefore are incapable to mediate apoptosis. Initial observations that TRAIL-R3 and TRAILR4 mRNA appeared to be primarily expressed on normal cells compared to tumour cells and that overexpression of TRAIL-R3 or TRAIL-R4 inhibited TRAIL-induced apoptosis, indicated that the relative expression of pro-apoptotic and decoy TRAIL-receptors could regulate the TRAIL sensitivity (Marsters et al., 1997; Pan et al., 1997a; Sheridan et al., 1997). However, subsequent studies using monoclonal antibodies specific for each receptor found no correlation between the levels of TRAIL-R3 or TRAIL-R4 and sensitivity of cells to TRAIL (Griffith et al., 1999; Wagner et al., 2007). It has been shown that TRAIL-R4 can suppress TRAIL-R2-mediated apoptosis by forming a ligand independent (Clancy et al., 2005) or ligand-dependent (Merino et al., 2006) complex. 2.3.4.5.2 Post-translational regulation of TRAIL-receptors Novel resistance mechanism involving expression of peptidyl O-glycosyltransferase GALNT14 was recently identified (Wagner et al., 2007). Loss of this enzyme correlated with reduced sensitivity to TRAIL because O-glycosylation of DR4 and DR5 promotes the ligand-stimulated clustering of receptors leading to more efficient recruitment and activation of caspase-8 (Wagner et al., 2007). 2.3.4.5.3 FLIPs Activation of caspase-8 and -10 can be regulated by cellular FLICE-inhibitory protein (c-FLIP). Several FLIP variants are generated by alternative splicing of the mRNA but only three of them are expressed at the protein level, the most abundant c-FLIP long (c-FLIPL), a c-FLIP short (c-FLIPS) and a short variant c-FLIPR. All of the isoforms have two DEDs and can be recruited to the DISC through DED-DED interactions. c-FLIPL contains caspase-like domain but lacks catalytic cysteine and therefore has no protease activity. The role of c-FLIPS in inhibiting death receptor-mediated apoptosis is well established. c-FLIPS was shown to block caspase-8 processing and activation at the DISC, probably competing for binding to FADD. As c-FLIPR structure closely resembles c-FLIPS, it is likely that these proteins inhibit death receptor-mediated apoptosis through similar mechanisms. On the contrary, the

34 function of c-FLIPL at the DISC remains controversial. Originally, c-FLIPL was described as an anti-apoptotic protein that inhibits death receptor induced apoptosis by interfering with caspase-8 activation at the DISC. Overexpression of c-FLIPL results in the recruitment of both c-FLIPL and procaspase-8 to the DISC, followed by defective processing of caspase-8. In addition, several studies have implicated c-FLIPL in the activation of the survival signaling pathways, especially NFB-pathway. On the contrary, some studies describe pro-apoptotic functions of c-FLIPL, referring to its assistance in the auto-catalytic activation of pro-caspase-8 at the DISC (Micheau et al., 2002). 2.3.4.5.4 Other mechanisms TRAIL-resistance can also result from reduced caspase-8 expression (Eramo et al., 2005; HopkinsDonaldson et al., 2000), increase expression of IAPs such as XIAP (Cummins et al., 2004) and cIAP2 (Ricci et al., 2007) or overexpression of anti-apoptotic Bcl-2 members including Bcl-2, BclXL and Mcl-1 (Fulda et al., 2002; Hinz et al., 2000; Ricci et al., 2007; Rosato et al., 2007). 2.3.4.6 Clinical trials of TRAIL Preclinical studies have established that using nontagged, soluble zinc-replete form of TRAIL is effective, nontoxic on various organs, including the liver, and, therefore suitable for clinical studies. As a result, soluble recombinant TRAIL (rh-APO2/TRAIL) and agonistic antibodies against TRAIL-R1 (mabatumumab or HGS-ERT1) or TRAIL-R2 receptors (lexatumumab or HGSERT2, AMG 655, apomab) are currently in PhaseI/II clinical trials for treatment of solid tumors and hematological malignancies (Johnstone et al., 2008). 2.4 Glucocorticoid-induced apoptosis 2.4.1 General Glucocorticoids (GCs) are a class of steroid hormones that exert a wide range of anti-inflammatory and immune-suppressive activities. Therefore, GCs such as dexamethasone are commonly used in the treatment of inflammatory and autoimmune diseases. In addition, the ability of GCs to induce cell cycle arrest and apoptosis in lymphoid cells, has led to their inclusion in chemotherapy protocols for lymphoid malignancies (Alexanian and Dimopoulos, 1994; Gaynon and Carrel, 1999; Moalli and Rosen, 1994).

35 2.4.2 Glucocorticoid receptor and function The effects of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the steroid hormone receptor superfamily that functions as ligand dependent transcription factor. GR consists of three distinct structural and functional domains. N-terminal region domain (NTD) contains a ligand independent transactivation domain. A central DNA binding domain consists of two highly conserved zinc finger domains and is essential for binding to glucocorticoid response element (GRE) sequences of regulated genes. This domain contains also nuclear localization sequence (NLS). The first zinc finger domain is necessary for binding to NF- B and AP-1 and for the transrepression function of the GR. The second zinc finger domain is involved in receptor dimerization and GRE transactivation. The C-terminal region contains the ligand binding domain which is also required for binding heat-shock proteins and GR dimerization. It also contains a ligand-dependent transactivation domain and nuclear localization signal (Frankfurt and Rosen, 2004). In its unactivated state, GR associates with heat shock proteins in the cytoplasm. Upon ligand binding, GR dissociates from the complex and translocates to the nucleus, and either increases or decreases gene expression (Herr et al., 2007).

Figure 6. Activation of GR.

Gene induction of GR is mediated via GR interaction with conserved GREs, whereas gene repression occurs through negative GREs, protein-protein interaction with other transcription factors, competition for co-activators and other mechanisms. GCs can also exert more immediate,

36 presumably non-genomic but still GR-dependent, effects. However these mechanisms are less well understood. In addition to the well-characterized cytoplasmic/nuclear GR, a membrane-associated GR was reported but its existence and possible significance remained controversial (Schmidt et al., 2004). GC-induced apoptosis is strictly dependent on the interaction of GC with its receptor. The requirement for the GR has been shown in thymocytes from genetically modified mice, and human acute lymphoblastic leukemia (ALL) cell lines with mutated GR and by conferring GC sensitivity to GC-resistant ALL-cell lines by GR transgenesis (Schmidt et al., 2004). Additionally, the level of GR expression is a critical determinant for GC sensitivity and GR autoinduction is required for GCinduced apoptosis (Kofler et al., 2003; Pedersen and Vedeckis, 2003; Ramdas et al., 1999; Riml et al., 2004). 2.4.3 Regulation of transcription specific genes It is widely accepted that GC-induced apoptosis results from alterations in gene expression. However, it is still controversial whether it requires gene transactivation, transrepression or both (Schmidt et al., 2004). Several studies have identified a large number (over 900) of genes during GC-induced apoptosis by using expression profiling and various model systems of GC-induced cell death (Schmidt et al., 2004). However distinct set of genes were regulated in different cell systems and experimental conditions indicating that multiple cell-context-dependent mechanisms rather than a conserved pathway may lead to GC-induced cell death. Some of these genes are directly implicated in death and survival decisions (such as Bim and granzyme A) or in cellular stress (such as I B

and c-myc). However, some of the genes are not causal in the death response.

Upregulation of GR itself was also shown (Schmidt et al., 2004). 2.4.4 Mechanisms of glucocorticoid-induced apoptosis GC has been shown to activate the apoptotic machinery by regulating components of either the extrinsic or intrinsic pathways or both. It has been shown that inhibition of caspase-8 prevented GC-induced release of cyt c and apoptosis (Marchetti et al., 2003). However, the involvement of death receptor pathway has been questioned and it has been suggested that caspase-8 is activated downstream from mitochondria since activation of caspase-8 was markedly reduced in Apafdeficient thymocytes treated with dexamethasone while death receptor-mediated activation of

37 caspase-8 was not diminished (Herr et al., 2007). Additionally, GC-induced apoptosis was unaffected in Bid-deficient mice (Yin et al., 1999). The involvelvement of the intrinsic pathway in GC-induced apoptosis is evident. In thymocytes from caspase-9 deficient mice GC-induced apoptosis and also the loss of mitochondrial membrane potential were prevented (Hakem et al., 1998; Kuida et al., 1998). In addition, it has been shown that dexamethasone induces loss of mitochondrial membrane potential in thymocytes and T cell hybridoma cells (Camilleri-Broet et al., 1998; Marchetti et al., 1996; Petit et al., 1995; Yoshino et al., 2001). Furthermore, overexpression of antiapoptotic Bcl-2 proteins attenuated GC-induced cell death in mouse thymocytes, human ALL and multiple myeloma cell lines (Feinman et al., 1999; Grillot et al., 1995; Hartmann et al., 1999). Since induction of proapoptotic proteins Bim (Abrams et al., 2004; Bachmann et al., 2005; Planey et al., 2003; Wang et al., 2003; Zhang and Insel, 2004) and Puma (Erlacher et al., 2005) as well as repression of antiapoptotic Bcl-2 and Bcl-XL (Casale et al., 2003; Chauhan et al., 2002) proteins have been observed in GC-treated cells, transcriptional regulation of Bcl-2 proteins may be essential for GC-induced apoptosis in many systems. 2.4.5 The role of lysosomes in GC-induced apoptosis Lysosomes may play an important role in both caspase-dependent and caspase-independent cell death (Kroemer and Jaattela, 2005). Central mediators in this process are cathepsins (such as cathepsin B), which are synthesized as proenzymes, transported into the lysosomal vesicle, and activated through proteolytic cleavage. As a result of lysosomal membrane permeabilization (LMP), cathepsin B is released from lysosomes to the cytosol, where it can induce cell death by activating initiator caspases or directly cleaving nuclear substrates (Kroemer and Jaattela, 2005). Cathepsin B activation was shown to be an early and essential step in the execution phase of GCinduced apoptosis in thymocytes. It was postulated that GC-induced activation of caspase-3 by caspase-9 in thymocytes occurs directly and also indirectly through a lysosomal amplification loop (Wang et al., 2006). 2.5 Regulation of apoptosis by PI3-kinase-Akt-pathway 2.5.1 General The phosphatidyl-inositol-3-kinase (PI3K)-Akt signaling pathway regulates fundamental cellular functions such as transcription, translation, proliferation, growth and survival. Disturbed activation

38 of PI3K-Akt pathway has been associated with development of cancer. Indeed, alterations in the PI3-kinase-Akt signaling pathway are frequent in human cancers. It has been suggested that constitutively active Akt promotes cellular survival and resistance to chemotherapy in solid tumors (Brognard et al., 2001; Clark et al., 2002; Ng et al., 2000; West et al., 2002) and in cells of hematopoietic lineage (Cataldi et al., 2001; Martelli et al., 2003; Mitsiades et al., 2002; O'Gorman et al., 2000). 2.5.2 Activation of PI3-kinase-Akt-pathway PI3K is a phospholipid-modifying enzyme that phosphorylates the phosphoinositols at the 3´ position of the inositol ring. It exists as a heterodimer of catalytic subunit (p110) and regulatory subunit (p85). Activation of the PI3-kinase signaling pathway involves recruiting PI3-kinase to the plasma membrane, where its substrates are located and tyrosine phosphorylation of PI3-kinase. Activation of PI3K leads to accumulation of two PI3-kinase products, PI(3,4)-P2 and PIP3. PI(3,4)P2 and PIP3 act directly as second messengers and can bind to and activate several protein kinases. PIP3 can also recruit cytoplasmic signaling proteins to the plasma membrane by binding to their pleckstrin homology (PH) domains (Franke, 2008). The downstream target of PI3-kinase pathway is serine/threonine kinase Akt, also called protein kinase B (PKB). Binding of PIP3 to the PH domain of Akt is important for recruiting Akt to the plasma membrane where it can be phosphorylated at two residues (Thr308 and Ser473) by upstream kinases and thereby activated (Franke, 2008). Negative regulation of the PI3-kinase-Akt pathway is mainly accomplished by phosphatase and tensin homologue (PTEN). PTEN is a dual-specificity phosphatase that has activity against lipid and protein substrates. The main physiological target of PTEN is PIP3, which is one of the PI3K products. PTEN reduces the amount of PIP3 and changes it to PIP2 through dephosphorylation of 3´ inositol position. Inactivating mutations or a loss of PTEN expression leads to increased activation of Akt followed by increased cell proliferation and resistance to apoptosis (Osaki et al., 2004). 2.5.3 Anti-apoptotic targets of Akt Activated Akt has several important functions including prevention of apoptosis. One way in which Akt may prevent apoptosis is by phosphorylating BH3-only protein Bad (at Ser-136). Phosphorylation of Bad creates a binding site for a member of the 14-3-3 protein family. When Bad is bound to 14-3-3, it cannot promote apoptosis (Zha et al., 1996).

39 Phosphorylation of forkhead family of transcription factors by Akt alters their intracellular localization. In the absence of Akt activation, FOXO proteins are predominantly localized in the nucleus where they are able to promote transcription of proapoptotic targets genes such Fas ligand, Bim and Puma (Brunet et al., 1999; Dijkers et al., 2000; You et al., 2006). When phosphorylated by Akt, FOXOs are exported from the nucleus and sequesterd by 14-3-3 proteins in the cytosol (Brunet et al., 1999). Akt has also been shown to directly phosphorylate procaspase-9 on Ser-196, and this phosphorylation correlates with the decrease in the protease activation of caspase-9 (Cardone et al., 1998). X-linked inhibitor of apoptosis protein (XIAP) has been suggested as potential Akt target since it has been shown that Akt phosphorylates and stabilizes XIAP (Dan et al., 2004). Akt can phosphorylate and activate I B kinase

(IKK ), which in turn phosphorylates I B,

targeting it for degradation (Kane et al., 1999; Ozes et al., 1999). This leads to nuclear translocation and activation of nuclear factor- B (NF- B). In addition, Akt phosphorylates and activates the cyclic AMP-response element-binding (CREB) protein, which increases the transcription of antiapoptotic genes, such as Bcl-2 and Mcl-1 (Du and Montminy, 1998; Pugazhenthi et al., 2000; Wang et al., 1999). Interestingly, Akt regulates indirectly tumor suppressor p53 protein, which acts as a cellular sensor of cellular stress and transduces stress signals into apoptotic signals. Akt promotes phosphorylation and nuclear translocation of Mdm2 (Hdm2 in humans) which destabilizes p53 leading to degradation of p53 (Mayo and Donner, 2001; Zhou et al., 2001).

Figure 7. Anti-apoptotic targets of Akt.

40 2.5.4 Glycogen synthase kinase-3 2.5.4.1 General Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase which is ubiquitously expressed in mammalian cells. Two GSK3 isoforms encoded by distinct genes have been identified, GSK3 and GSK3 . Initially, GSK3 was identified as a kinase that phosphorylates and inactivates a key metabolic enzyme, glycogen synthase. Subsequently GSK3 has been shown to be involved also in several other cellular processes, including apoptosis. Interestingly, GSK3 has the capacity to either increase or decrease the threshold for apoptosis (Beurel and Jope, 2006). 2.5.4.2 Regulation of glycogen synthase kinase-3 activity The activity of GSK3 is tightly controlled by multiple mechanisms. Phosphorylation of Ser-21 in GSK3- or Ser-9 in GSK3- inhibits its activity. PI3-kinase-Akt signaling pathway is a major regulator of GSK3 because Akt phosphorylates GSK3 on these serine residues. However, several other kinases have also been shown to phosphorylate them. Conversely, the enzymatic activity is enhanced by phosphorylation of tyrosine-279 in GSK3- or tyrosine-216 in GSK3- . However, the mechanisms regulating these phosphorylations are not well-defined. In addition to phosphorylation, GSK3 activity can be regulated by complex formation and by the control of its intracellular localization. Additionally, the action of GSK-3 is usually indirectly controlled by the phosphorylation state of its substrate since the most substrates of GSK-3 must be prephosphorylated (Beurel and Jope, 2006). 2.5.4.3 GSK3 promotes intrinsic apoptosis GSK3 has been shown to promote mitochondrial intrinsic apoptosis. Overexpression of GSK3 was found to induce apoptosis (Pap and Cooper, 1998). After that, activation of GSK-3 has been shown to promote apoptosis induced by various stimuli, including staurosporine (Bijur et al., 2000), inhibition of PI3-kinase (Pap and Cooper, 1998), serum deprivation (Linseman et al., 2004), mitochondrial toxins (King et al., 2001) and DNA damage (Beurel et al., 2004; Beurel et al., 2005; Jin et al., 2005; Tan et al., 2006; Watcharasit et al., 2002; Watcharasit et al., 2003). It has been shown that GSK3 is present in mitochondria and mitochondrial GSK3 activity is increased during intrinsic apoptotic signaling induced by DNA damage or ER stress (Bijur and Jope, 2003). Direct

41 mitochondrial substrates have not yet been found. However, studies have shown that its apoptotic action is upstream of caspase-9 (Bijur et al., 2000), cytochrome c release from mitochondria (Watcharasit et al., 2003) and PTP complex in mitochondria (Juhaszova et al., 2004). In addition, GSK3 can directly phosphorylate Bax, which results in activation of Bax (Linseman et al., 2004). Phosphorylation by GSK3 enhances degradation of Mcl-1 (Maurer et al., 2006). GSK3 also phosphorylates VDAC which prevents hexokinase II from associating with VDAC which may faciliate the mitochondrial association of pro-apoptotic Bcl-2 family members to promote apoptosis (Cheng et al., 2003; Majewski et al., 2004; Pastorino et al., 2002). Mitochondrial function during intrinsic apoptosis signaling is also influenced by GSK3-mediated regulation of the expression of proteins participating in the mitochondrial stage of apoptosis. GSK3 regulates in the nucleus transcription factors p53 and CREB. GSK3 activity promotes p53-induced expression of Bax in response to DNA damage (Tan et al., 2005; Watcharasit et al., 2003) and inhibits CREB-dependent expression of Bcl-2 (Bullock and Habener, 1998; Grimes and Jope, 2001). GSK3 has also been shown to be required for stress-induced expression of Bim (Hongisto et al., 2003). 2.5.4.4 GSK3 inhibits extrinsic apoptosis GSK3 has an opposite effect on extrinsic apoptosis inhibiting the death-receptor-mediated apoptosis. It has been shown in human prostate cancer cell lines that lithium and another highly selective inhibitor of GSK3, SB216763, as well as by knockdown of the GSK3 protein level using RNA interference potentiates TRAIL-induced apoptosis (Liao et al., 2003). Apoptosis induced by stimulation of DR5 with an agonistic antibody was shown to be potentiated by inhibition of GSK3 (Rottmann et al., 2005). In addition, apoptosis induced by Fas was found to be regulated by the anti-apoptotic action of GSK3 since several inhibitors of GSK3 potentiated Fas-induced apoptosis in Jurkat cells and in neurons (Song et al., 2004). Several studies have shown that inhibition of GSK3 enhances the death receptor apoptosis already upstream of the activation of caspase-8 (AzaBlanc et al., 2003; Liao et al., 2003; Schwabe and Brenner, 2002). The precise mechanism remains to be identified.

42 3. AIMS OF THE STUDY The overall aim of this study was to investigate detailed molecular mechanisms and regulation of mitochondrial and death receptor apoptotic pathways in human FL cell lines. Detailed knowledge of the regulatory mechanisms of the apoptotic pathways is necessary for development of novel therapeutic strategies for FL. In addition, as germinal center microenvironment of FL cells, especially CD40 signaling, could have an impact on the treatment, the effect of CD40 signaling on TRAIL- and drug-induced apoptosis was evaluated.

The specific aims were 1.

To examine the detailed molecular mechanisms of dexamethasone (Dex)-induced apoptosis and the involvement of known survival pathways in the regulation of Dexinduced apoptosis in human follicular lymphoma cell line (I).

2.

To elucidate the requirement of mitochondria and the role of glycogen synthase kinase-3 (GSK3) in dexamethasone-induced apoptosis (II).

3.

To determine type I and type II signaling pathways in TRAIL-induced apoptosis (III).

4.

To investigate whether the CD40-CD40L interactions between FL cells and the cells in their GC microenvironment could have an impact on the treatment of FL and to elucidate how the possible anti-apoptotic function of CD40 could be counteracted (IV).

43 4. MATERIALS AND METHODS 4.1 Cell lines and culture conditions (I-IV) All the experiments have been performed on human follicular lymphoma cell lines HF1A3, HF28RA and HF4.9 (Eray et al., 2003; Matto et al., 2005). The cell lines have been established from enlarged lymph nodes of FL patients. The origin and characteristics of the cell lines have been described previously (Eray et al., 2003). As a hallmark of a FL, all three cell lines carry the t(14;18) translocation and overexpress Bcl-2 protein (Eray et al., 2003). Cells were cultured at 37 C in a moist atmosphere of 5 % CO2 in RPMI 1640 media (Gibco BRL Life Sciences, Paisley, Scotland) supplemented with 5 % heat-inactivated fetal calf serum (FCS, Biological Industries, Kibbuz Beit Haemek, Israel), 2 mM L-glutamine, 200 µg/ml streptomycin (Sigma, Stenheim, Germany), 240 IU/ml penicillin (Orion, Espoo, Finland), 10 mM HEPES buffer (Gibco BRL), 0.1 mM nonessential amino acids (Gibco BRL), 1.0 mM sodium pyruvate (Gibco BRL), and 20 µM 2-mercaptoethanol (Fluka Chemie, Buch, Switzerland). 4.2 Cell treatments (I-IV) Dexamethasone was from Sigma-Aldrich (Stenheim, Germany) (I, II, IV). Inhibitors of signaling pathways PI3-kinase inhibitor LY294002, Akt-inhibitor (1L-6-hydroxy-methyl-chiro-inositol 2(R)2-O-methyl-3-O-octadecylcarbonate), MEK inhibitor PD98059, protein kinase C (PKC) inhibitor Gö6850 and Raf Kinase Inhibitor ZM336372, were purchased from Calbiochem (La Jolla, CA) (I). Protein synthesis inhibitor cycloheximide (CHX) was purchased from Sigma-Aldrich (I). SB216763 (Sigma-Aldrich) and LiCl (Sigma-Aldrich) were used to inhibit GSK3 (II). His-tagged recombinant human soluble Killer TRAIL

was obtained from Apotech (Epalinges, Switzerland)

(III, IV). Doxorubicin hydrochloride solution was obtained from Sigma-Aldrich (IV). Pyrrolidinedithiocarbamate (PDTC) (Sigma-Aldrich) and IKK inhibitor III (BMS-345541) (Calbiochem, La Jolla, CA) were used to inhibit NF- B pathway (II, IV). The anti-CD40 mouse mAb AF1.15 was gently provided by Dr Matti Kaartinen from Haartman institute, University of Helsinki, Finland (IV). Anti-TRAIL-R1 (clone HS101) and anti-TRAIL-R2 (clone HS201) antibodies (Abs) (Alexis Biochemicals, Gruenberg, Germany) were used for FACS staining and receptor blockade.

44 4.3 3H-thymidine incorporation Thymidine incorporation was carried out using 6 x 10E4 cells/well in 200 µl of medium containing appropriate stimuli. After 20 h of incubation, 3µCi of (3H) thymidine (specific activity 2.0 Ci/mmol, Amersham Biosciences, Piscataway, NJ) was added to each well for 4 h. Cells were harvested and the incorporated radioactivity was detected by beta scintillation counting. 4.4 Propidium iodide staining (I-IV) DNA fragmentation was detected by flow cytometric analysis after propidium iodide staining. Cells with fragmented DNA were considered as apoptotic. Fixation and staining of the cells were performed according to standard protocols. Briefly, samples containing 1 x 106 cells were collected, resuspended in ice-cold PBS and fixed with ethanol (70 % v/v). After overnight incubation at +4 C, cells were centrifuged and incubated with 150 g/ml RNase for 1 h at 50 C. Propidium iodide (PI) (Molecular Probes, Leiden, Netherlands) was added to the final concentration of 8 g/ml and incubation was continued for 2 h at +37 C. FACScan flow cytometer with CellQuest v 3.1 software (Becton Dickinson, Mountain View, CA) was used in the analysis. 4.5 Enzyme assay for caspase activity (I) Activity of caspase-3 was analyzed by measuring the cleavage rate of a synthetic fluorescent substrate Ac-DEVD-AMC (Calbiochem). The protease assay was carried out according to the manufacturer’s instructions. Briefly, 2 x 106 cells per sample were lyzed in 100 l of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 10mM NaH2HPO4, 10mM Na2HPO4, 1% Nonidet P-40, 0.1 mM PMSF, 1 M VO4, 5 g/ml aprotinin, 5 g/ml leupeptin). After 30 min of incubation on ice, the cell lysate was centrifuged (10,000

g, 30 min) and the supernatant was used

as a cytosolic extract. The concentration of cytosolic proteins was measured by Lowry’s method (DC protein assay, Bio-Rad laboratories, Hercules, CA). Equal amount of protein was dissolved in protease assay buffer (20 mM Hepes, 10 % glycerol, 2mM DTT). Samples were incubated with 20 M Ac-DEVD-AMC fluorogenic substrate for 2 h. After incubation, released fluorogenic AMC was measured using Wallac Victor 1420 spectrofluorometer (Wallac, Turku, Finland) with an excitation wavelength of 355 nm and emission wavelength of 460 nm.

45 4.6 Detection of changes in mitochondrial membrane potential (

m)

4.6.1 ApoAlert Mitochondrial Membrane Sensor Kit (I) ApoAlert Mitochondrial Membrane Sensor Kit (Clontech Laboratories, Inc., Palo Alto, CA) was used for detecting changes in TM

MitoSensor

m

during apoptosis. The kit contains a cationic dye

that fluoresces differently in apoptotic and non-apoptotic cells, because of changes

in mitochondrial membrane permeability. Cells were stained and analyzed according to manufacturer’s instructions. Briefly, 5 x 105 cells were collected and resuspended in 500 l of the incubation buffer containing 0.5 l MitoSensor TM reagent and incubated for 15 min at 37 C in a 5 % CO2 incubator. After staining cells were washed with and resuspended in the Hepes buffer (10 mM Hepes, 140 mM NaCl, 5 mM CaCl2, pH 7.4) and the fluorescence signal was analyzed by flow cytometry. Forward and side scatter was used for the selection of early apoptotic cells. 4.6.2 TMRM staining (II, III) Depolarization of mitochondrial membrane was detected by TMRM staining. TMRM is the methyl ester of tetramethylrhodamine and it is detected in FL2 channel. After incubation of appropriate stimuli, cells (1x10E6) were stained with 150 nM TMRM (Molecular Probes) for 15 min at 37 °C in the dark. After staining cells were immediately analyzed with FACScan flow cytometer (Becton Dickinson). Forward and side scatter were used to gate living/early apoptotic cells for TMRM analysis. 4.7 FACS analysis of receptors Cells (5x105) were incubated with mAbs against TRAILR1 and TRAILR2 at the concentration of 1µg in 100 µl of FACS-buffer (2% FCS in PBS) followed by PE-labeled goat anti-mouse (BD Pharmingen) at the concentration of 0.4 µg/ml. Stained cells were analyzed using FACScan flow cytometry equipped with a CellQuest data analysis program (Becton Dickinson, San Diego, CA, USA). Isotype matched (mouse IgG1) antibody was used to control the unspecific binding of antibodies.

46 4.8 Preparation of samples for Western blot analysis (I, III, IV) After stimulation, cells (2

106) were collected and resuspended with lysis buffer (20 mM

Tris/HCl (pH 8.0), 2 mM EDTA, 3% NP-40, 100 mM NaCl, 50 mM NaF, 1 M PMSF, 1 M VO4, 5 g/ml aprotinin, 5 g/ml leupeptin). After 30 min incubation on ice samples were centifuged at 10 000 g, 15 min, + 4 C. Protein concentration of the samples was measured and equalized with SDS-PAGE buffer (0.125 M Tris-Cl pH 6.8, 4 % SDS, 20 % glyserol, 10 % 2-mercaptoethanol, bromofenolblue). 4.9 Preparation of mitochondrial and cytosolic fractions (I-III) Mitochondrial and cytosolic fractions were obtained using ApoAlertTM cell fractionation kit (Clontech Laboratories) according to the manufacturer’s protocol. In brief, 2

107 cells were

stimulated harvested and washed with wash buffer. The cell pellet was resuspended in 0.8 ml fractionation buffer containing protease inhibitors and DTT, and incubated on ice for 10 min. Subsequently, cells were homogenized by passing the cell suspension through a 27G syringe needle. The homogenate was centrifuged at 700 was centrifuged at 10,000

g for 10 min at 4 C. The remaining supernatant

g for 25 min at 4 C. The supernatant (a cytosolic fraction) was

collected and the pellet (mitochondrial fraction) was resuspended in 0.1 ml of the fractionation buffer. The protein concentration of fractions was determined and equalized with SDS-PAGE buffer. 4.10 Western blot analysis (I-IV) Equal amount of protein from each sample was separated on 12 %-15% SDS-PAGE gel and transferred to nitrocellulose membranes (Hybond ECL nitrocellulose membranes, Amersham Biosciences). Membranes were blocked overnight or for 1h in PBS containing 3% BSA and 0.1% Tween or 5 % milk and 0.1% Tween at room temperature and incubated for 2 h with primary antibodies. After washing, the membranes were incubated for 30 min-1 h with horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat or rabbit anti-mouse antibodies (Zymed Laboratories Inc, South San Fransisco, CA). Enhanced chemiluminescence (ECL) system (Amersham Biosciences) was used for detection.

47 4.10.1 Antibodies (I-IV) The antibodies used in immunoblot analysis were rabbit polyclonal anti-cytochrome c (Clontech) (I,II,III), polyclonal anti-Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA) (I), mouse monoclonal anti-Bad (Transduction Laboratories, Lexington, KY) (I), mouse monoclonal anti-Bax (Transduction Laboratories) (I), rabbit polyclonal anti-Bcl-2 (Santa Cruz) (I), goat polyclonal antiAkt1/2 (Santa Cruz) (I), rabbit polyclonal anti-phospho-Akt (Ser-473) (Santa Cruz) (I), rabbit polyclonal anti-Bim (Sigma-Aldrich) (I,II), mouse monoclonal anti-caspase-8 (Cell Signaling technology, Beverly, USA) (III), rabbit polyclonal anti-caspase-9 (H-83) (Santa Cruz) (III), mouse monoclonal anti-Bcl-XL (2H12) (Zymed laboratories Inc., CA, USA) (III, IV), rabbit polyclonal anti-Bid (Cell signaling technology) (III), rabbit polyclonal anti-FLIP S/L (H-202) (Santa Cruz) (IV), rabbit polyclonal anti-XIAP (ProSci Inc., Poway, CA) (IV), rabbit polyclonal anti-cIAP (ProSci Inc.) (IV), rabbit polyclonal anti-phospho-GSK3a/b (Ser21/9) (Cell Signaling technology) (II), rabbit polyclonal anti-caspase-3 (Santa Cruz), mouse monoclonal anti-cyclin D3 (BD Transduction Laboratories), mouse monoclonal Kip1/p27 (BD Transduction Laboratories), mouse monoclonal anti-COX4 (Clontech) (II), rabbit polyclonal anti-actin (SantaCruz) (II, III), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.), HRPrabbit anti-goat IgG (Zymed Laboratories Inc.) and HRP-goat anti-mouse IgG (Zymed Laboratories Inc.). 4.11 RT-PCR (IV) Briefly 1x106 cells were washed twice with cold PBS and the pellets were stored at -70 C prior to analysis. Total mRNA extraction was done by using MasterPureTM RNA purification Kit (Epicentre Technologies, WI) according to manufacturer’s instructions. Total mRNA amounts were measured using spectrophotometry (GeneQuant, Pharmacia). For cDNA sythesis 2 µg mRNA, 40 pmol p(dT)15 and RNA water ad 18 µl were mixed and incubated at 65 C for 5 min. After incubation 1 X RT buffer (5 X M-MLV RT buffer, Promega), 10 mM each dNTP and 8 U of RT (M-MLV RT, Promega) were added to reaction mixture (final volume 25 µl). cDNA was synthesized at 45 C for 1 h. After synthesis 1:1 TE buffer was added and samples were boiled for 3 min. cDNA synthesis was controlled by PCR using

2µ-globulin

primers (sense 5'-CCA-GCA-GAG-AAT-GGA-AAG-TC-3', anti-sense 5'-GAT-GCT-GCT-TACATG-TCT-CG-3'). Other primers used were as follows: FLIP-S sense 5`-CTG-GTT-GCC-CCA-

48 GAT-CAA-CTG-3`, FLIP-S anti-sense 5`CTG-GTT-GCC-CCA-GAT-CAA-CTG-3`, FLIP-L sense 5´-GTA-TAT-CCC-AGA-TTC-TTG-GC-3`, FLIP-L anti-sense 5`GGC-TTC-CCT-GCTAGA-TAA-GG-3`, BCL-XL sense 5'-ATG-GCA-GCA-GTA-AAG-CAA-G-3', BCL-XL anti-sense 5'-GCT-GCA-TTG-TTC-CCA-TAG-A-3', C-IAP-1 sense 5'-GCC-ATC-TAG-TGT-TCC-AGTTC-3', C-IAP-1 anti-sense 5'-CAG-TGG-TAT-CTG-AAG-TTG-AC-3', C-IAP-2 sense 5'-CAATTG-GGA-ACC-GAA-GGA-TA-3', C-IAP-2 anti-sense 5'-ACT-TGC-AAG-CTG-CTC-AGG-AT3', XIAP sense 5’-CGA-AGT-GAA-TCT-GAT-GCT-GTG-3’, XIAP anti-sense 5’-CCC-TCCTCC-ACA-GTG-AAA-GC-3’,

survivin

sense

5'-CTG-AGA-ACG-AGC-CAG-ACT-TG-3',

survivin anti-sense 5'-GCA-CTT-TCT-TCG-CAG-TTT-TC-3'. Polymerase chain reaction (PCR) was carried out under the following conditions: 3 min at 94 C followed by 35 cycles of 1 min at 94 C, 1 min at 57 C and 1 min at 72 C with a final 10 min extension at 72 C. PCR products were separated by agarose gel electrophoresis. 4.12 Preparation of nuclear extracts (IV) For the preparation of nuclear extracts the cells were washed with PBS and suspended in a hypotonic buffer (1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 mM HEPES). After 5 min incubation on ice, 0.08% NP-40 was added and suspension was incubated further 2 min before centrifugation (400 g, 2 min, 4 C). Pellets containing the nuclei were suspended in hypertonic buffer (25 % glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 420 mM NaCl, 20 mM HEPES) and incubated at 4 C for 30 min with smooth shaking. Finally, samples were centrifuged (25 000 g, 20 min, 4 C) and supernatants were stored at -80 C. The amount of nuclear proteins was analyzed with Lowry's method (DC protein assay, BioRad Laboratories). 4.13 EMSA assay (IV) Double-stranded oligonucleotide containing the NF- B consensus binding (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega, Madison, WI, USA) was labeled with [ -32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech Benelux, AT Roosendal, Netherlands) using T4polynucleotide kinase (MBI Fermentas, Hanover, MD, USA). Labeled probe was separated using Probe Quant G-50 micro columns (Amersham Pharmacia Biotech Benelux) prior to their use in EMSA experiments.

49 Extracted nuclear proteins (6 µg protein per reaction) were incubated for 20 minutes with [ 32

P]ATP labeled probes in binding buffer (10 % glycerol, 1 mM DTT, 1 mM EDTA, 25 mM

HEPES, 100 mM NaCl) and 1.5 µg poly(dI-dC) in final reaction volume of 20 µl. The proteinDNA complexes were separated (25 mA, 90-120 min) on a high ionic strenght gel (6 % acrylamide) in running buffer (50 mM Tris, 380 mM glycine, 1 mM EDTA). After electrophoresis, the gel was dried (1h, +60 C) and exposed to autoradiography film for 1-2 days at -80 C. 4.14 Cloning (II-IV) Plasmids coding for caspase-9, caspase-10, Bcl-XL, dominant negative-FADD, FLIP-S and FLIPL, were obtained by amplification of caspase-9, caspase-10, Bcl-XL, DN-FADD (lacking the death effector domain (DED)) sequences by PCR using cDNA from HF28RA as template. Primer sequences for amplification were for caspase-9 (S 5'-AT ATT TAA ATA GCC ACC ATG GAC GAA GCG GAT CGG-3', AS 5'-ATA TTT AAA TGG GCC CTG GCC TTA TGA TGT-3'), caspase-10 (S 5'-AT ATT TAA ATA GCC ACC ATG AAA TCT CAA GGT CAA C -3', AS 5'-A TAT TTA AAT CTG CTA TAA TGA AAG TGC ATC-3'), Bcl-XL (S 5'-AT ATT TAA ATA GCC ACC ATG TCT CAG AGC AAC GGG AGC-3', AS 5'-ATA TTT AAA TCA GTG TCT GGT CAT TTC CGA C-3'), FADD (S 5-AT ATT TAA ATA GCC ACC ATG CGC GTC GAC GAC TTC GAG GCG G-3’, AS 5'-AT ATT TAA ATG CCC ATC AGG ACG CTT CGG AG3’), FLIP-S (S 5'-ATA TTT AAA TCT AAG AGT AGG ATG TCT GCT GAA G-3', AS 5'-ATA TTT AAA TAT GTT AAT CAC ATG GAA CAA TTT C-3') and FLIP-L (S 5'-ATA TTT AAA TCT AAG AGT AGG ATG TCT GCT GAA G-3', AS 5'-ATA TTT AAA TGG TTT CTT ATG TGT AGA GAG GAT AAG-3'). Swa I- restriction enzyme cleavage sites (ATTTAAAT) were designed to the end of primers (bolded). PCR fragments were isolated from 1.2% agarose gel using Montage kit (Millipore) and then sub-cloned into the pcr2.1-TOPO-TA vector (Invitrogen, Carlsbad, CA) and transformed to competent One Shot TOP10-E. coli (Invitrogen). 4.15 Site-directed mutagenesis, cloning and production of lentiviruses (II-IV) Dominant negative caspase-9 and caspase-10 were generated by introducing a point mutation to catalytic cysteines, Cys287Ser (TGT AGT) and Cys401Ser (TGC AGC), respectively. Point mutation was generated by megaprimer method. Megaprimers were generated by PCR using Phusion DNA polymerase (Finnzymes, Espoo, Finland). Mutation primer for caspase-9 was S 5'-C CAG GCC AGT GGT GGG GAG CAG-3' and for caspase-10 S 5'- C ATC CAG GCC AGC CAA

50 GGT GAA G-3' (point mutations are bolded in primer sequence) and these primers were pared with SwaI-sites containing anti-sense primers mentioned above. PCR products were isolated from agarose gel and used as megaprimer in second round PCR with SwaI-sites containing sense primers mentioned above. PCR products were isolated from agarose gel and then sub-cloned into the pcr2.1-TOPO-TA vector and transformed to competent One Shot TOP10-E.coli. Inserts in TOPO-vectors were fully sequenced and verified to contain the desired product. After sequencing inserts were cloned to Swa I site of the pWPI-IRES-GFP (TronoLab, Switzerland) lentivirus vector. The lentiviral vectors containing DN-caspase-9-IRES-GFP, DN-caspase-10-IRES-GFP, DN-FADD-IRES-GFP and BclXL-IRES-GFP were produced following the protocol described earlier [41]. IRES-GFP pWPI lentiviruses were also produced and used as vector control. Titers of produced lentiviruses were 1.4x10E6 t.u.(transducing units)/ml (Bcl-XL), 4.3x10E6 t.u./ml (DN-casp9), 2.8x10E7 t.u./ml (DNcasp10), 8.3x10E6 t.u./ml (DN-FADD) and 5.8x10E7 t.u./ml (vector control) as measured by FACS analysis [41]. 4.16 Lentiviral transduction, FACS analysis and sorting of GFP-positive cells (II-IV) The HF28RA and HF1A3 cells were transduced with lentiviral vectors in 6-well plate using 100000 cells/ml/well, in the presence of polybrene 8 g/ml (SigmaAldrich, Stenheim, Germany) and with 1 t.u./cell. After three days, cells were collected and cultured in fresh medium and the amount of GFP-positive cells were analyzed by FACScan flow cytometer and after expanding the cells, the transduced cells were further enriched and purified over 96 % pure population, according to the GFP expression with cell sorting using EPICS Elite ESP flow cytometer (Beckman Coulter, Fullerton, CA, USA).

51 5. RESULTS AND DISCUSSION 5.1 Dexamethasone induces cell cycle arrest and apoptosis in dose-dependent manner (unpublished data) Glucocorticoids (GCs) have been shown to induce cell cycle arrest and apoptosis in lymphoid cells (Ausserlechner et al., 2004; Gaynon and Carrel, 1999; King and Cidlowski, 1998; Renner et al., 2003). Synthetic glucocorticoid, dexamethasone (Dex), induced cell cycle arrest and apoptosis in a dose-dependent manner in HF28RA cells (Fig.8). Based on thymidine incorporation, proliferation of HF28RA cells decreased considerably at the concentration of 0.01 µM (Fig.8A) whereas apoptosis was induced at ten-time higher concentration of Dex as demonstrated by flow cytometric analysis after PI-staining (Fig.8B). The decrease in the proliferation after Dex (0.01 µM) treatment was not due to the apoptotic cell death, but rather due to the induction of cell cycle arrest at G0/G1 (Fig.8C). Furthermore, HF28RA cells induced to G0/G1 cell cycle arrest for 24 h could enter back to the cycle within the next 24 h (data not shown). Other studies have also indicated that anti-proliferative effects of GCs may be mediated by a reversible G1-block in cell cycle progression since after release of Dex, cells reinitiated cell cycle progression and entered S-phase (Glick et al., 2000; Goya et al., 1993; Sanchez et al., 1993). In addition, prolonged induction of cell cycle arrest (48 hours) did not lead to apoptosis and overexpression of Bcl-XL prevented Dex-induced apoptosis (discussed later, II, Fig.1) but not cell cycle arrest in HF28RA cells (data not shown). It was also shown in human ALL cells that Bcl-2 protected cells against GC-induced apoptosis but did not affect GC-mediated growth arrest separating the anti-proliferative and apoptosis-inducing effects of GCs (Hartmann et al., 1999). Thus, our finding supports the previous suggestion that cell cycle arrest and apoptosis are not directly connected to each other, although it has been thought to be the case in highly proliferating cancer cells (King and Cidlowski, 1998).

52

A)

B)

C) Control

Hypodiploid G0/G1 S/G2/M

Dex 0.01 µM

Control 4.08 56.52 39.11

% of cells Dex 0.01µM 8.74 84.94 6.24

Figure 8. Dex induces apoptosis and cell cycle arrest in a dose dependent manner in HF28RA cells. (A) Cells were treated with indicated concentrations of Dex. After 20 h of incubation, 3µCi of (3H) thymidine was added to cells for 4 h. Cells were harvested and the incorporated radioactivity was measured by beta scintillation counting. (B,C) Cells were incubated in indicated concentrations of Dex. After 24 h of incubation, cells were harvested for cell cycle analysis. Hypodiploid cells (apoptotic cells), cells at G0/G1 and cells at S/G2/M are indicated as histogram regions M1, M2 and M3, respectively. The proportion of cells at different stages of cell cycle is shown under the histograms.

Cell cycle progression is controlled by cyclin-dependent kinases (CDKs) and their regulatory subunits, cyclins. The activity of cyclin D-dependent kinases CDK4 and CDK6 is tightly regulated

53 by inhibitors, including p27Kip. They halt cell cycle at G1 phase, by binding to, and inactivating, cyclin-CDK complexes. Active CDK4/6 complexes phosphorylate the retinoblastoma protein (Rb). The hyperphosphorylation of Rb leads to the release and activation of E2F, the transcription factor essential for the progression of cell cycle from G1 to S (Ausserlechner et al., 2004). Cell cycle regulators shown to be modulated by GCs include cyclin D3, c-myc, CDK4 and CDK6, which have been shown to be downregulated as well as p21Waf1 and p27Kip which have been shown to be upregulated (Ausserlechner et al., 2004; Greenstein et al., 2002). Our results also showed that Dexinduced downregulation of cyclin D3 in 8h and slight upregulation of p27Kip in 16 h as demonstrated by Western blotting (Fig.9).

Control Dex 1h 1h

Dex 4h

Dex 8h

Dex Control 16 h 16 h

CyclinD3 p27Kip

Figure 9. Dexamethasone decreases the amount of cyclin D3 protein and slightly increases the amount of p27Kip. Cells were treated with 0.01 µM Dex for 1, 4, 8 and 16 h and the amount of cyclin D3 and p27Kip proteins were detected by Western blotting.

5.2 Mechanism of Dex-induced apoptosis (I, II) GCs are frequently used in the treatment of various lymphoid malignancies, including leukemia, lymphoma and multiple myeloma. Despite their extensive clinical use, the molecular mechanisms leading to GC-induced apoptosis are not fully understood. In order to better understand the molecular mechanisms of Dex-induced apoptosis we analyzed the kinetics and the signaling requirements of Dex-induced apoptosis in a human follicular lymphoma cell line, HF28RA. Dex induced apoptosis in a delayed manner. The apoptotic changes including the loss of mitochondrial membrane potential (

m),

release of cyt c from mitochondria to cytosol, activation of caspase-3

(measured as protease activity against a fluorogenic DEVD peptide) and DNA fragmentation were induced after 12 h stimulation with Dex (I, Fig.1 and 6C). Kinetics of caspase-3 activation was also analyzed by Western blotting and similar results were obtained (Fig.10). Caspase-8 was also activated as demonstrated by Western blotting (Fig.10).

54 Involvement of mitochondrial changes in Dex-mediated apoptosis has also been demonstrated in several other studies with many cell types, including lymphoid cells (Camilleri-Broet et al., 1998; Castedo et al., 1995; Herr et al., 2007). In addition, we showed that overexpression of Bcl-XL completely prevented Dex-induced apoptotic changes indicating that mitochondrial activation is necessary for Dex-induced apoptosis (II, Fig.1C, D). Other studies have also shown that overexpression of anti-apoptotic Bcl-2 family proteins protects against GC-induced apoptosis (Alnemri et al., 1992; Caron-Leslie et al., 1994; Hartmann et al., 1999; Smets et al., 1999). In addition, GC-induced cell death was enhanced in thymocytes from Bcl-2 knockout mice (Veis et al., 1993). It is commonly thought that MOMP results in the release of cyt c from mitochondria leading to the formation of apoptosome and subsequent activation of caspase-9. Therefore, it is not surprising that overexpression of dominant negative (DN) caspase-9 prevented Dex-induced apoptosis (II, Fig.1E). Other studies have also shown that caspase-9 is necessary for GC-induced apoptosis (Hakem et al., 1998; Kuida et al., 1998; Planey et al., 2003). Interestingly, DN caspase-9 prevented also the loss of

m

(II, Fig.1F) indicating that caspase-9 is acting upstream of or even within mitochondria or

serves an essential amplification loop back to mitochondria. Previous studies have also shown that caspase-9 regulates mitochondrial changes. It was shown that caspase-9 deficiency in caspase-9 knockout mice prevented Dex-induced loss of mitochondrial membrane potential (Hakem et al., 1998; Kuida et al., 1998). In addition, the rituximab-induced release of cytochrome c and loss of mitochondrial membrane potential were regulated by caspase-9 in FL cells (Eeva et al., 2009). It has also been shown in caspase-3 and caspase-7 double knockout mice that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis (Lakhani et al., 2006). The detailed mechanisms how caspases regulate mitochondrial events are not known but I would like to speculate that there is an essential amplification loop from caspase-9 back to mitochondria via caspase-3(/7) and caspase-8 since activation of caspase-8 was also induced by Dex (Fig.10). Recently, it was shown that cathepsin B was released from lysosomes to the cytosol in thymocytes after GC treatment (Wang et al., 2006). It was suggested that caspase-9 activates directly caspase-3 and also indirectly through lysosomal amplification loop by activating the release of cathepsin B from lysosomes to cytosol leading to activation of caspase-8 and subsequently activation of caspase-3 (Wang et al., 2006). Future studies will show whether there is co-operation of mitochondrial and lysosomal death pathways during Dex-induced apoptosis in HF28RA cells.

55 It has been previously reported that Dex-induced apoptosis is countered by protein synthesis inhibitors (Cifone et al., 1999; Lemaire et al., 1999). The slow kinetics of Dex-induced apoptosis offers plenty of time for protein synthesis to occur. Indeed, in our experiments, treatment of HF28RA cells with protein synthesis inhibitor, cycloheximide (CHX), prevented partly Dexinduced apoptosis in a dose dependent manner (I, Fig.2A), indicating that new protein synthesis is required for the induction of apoptosis. In addition, our results showed that protein synthesis is required already before or at mitochondrial stage of apoptosis (I, Fig.2B). However, the inability of CHX to completely prevent Dex-induced apoptosis indicates that protein synthesis independent apoptotic mechanisms could also be involved. However, more probable explanation could be that the amount of CHX needed to prevent Dex-induced protein synthesis becomes toxic itself. The Bcl-2 family member consistently shown to be upregulated by glucocorticoids in lymphoid cells is Bim (Abrams et al., 2004; Bachmann et al., 2005; Planey et al., 2003; Wang et al., 2003; Zhang and Insel, 2004). We showed that Dex induced upregulation of all three major isoforms of Bim in HF28RA cells. Upregulation of Bim was shown from total cell lysates (I, Fig.2C) and also from mitochondrial fraction of cellular proteins (I, Fig.7). The three major isoforms of Bim (BimEL, BimL and BimS) are generated by alternative splicing and all of them induce apoptosis. The kinetics of the apoptotic events correlated with the upregulation of the Bim protein indicating that Bim plays a central role in Dex-induced apoptosis in HF28RA cells. The ratio of proapoptotic to anti-apoptotic Bcl-2 family members is thought to be critical in determining whether the cell will undergo apoptosis. HF28RA cells, as most cases of follicular lymphoma (Weiss et al., 1987), have a t(14;18) translocation, which causes over-expression of the bcl-2 gene (Eray et al., 2003). In some cases a high level of Bcl-2 expression has been shown to delay, but not to prevent GC-induced apoptosis (Hartmann et al., 1999; Strasser et al., 1991; Strasser et al., 1995). Therefore, one explanation for slow kinetics of Dex-induced apoptosis could be that accumulation of Bim protein in mitochondria is required to antagonize first the antiapoptotic Bcl-2 (and also other anti-apoptotic Bcl-2 family members) before Bax and/or Bak are activated to induce apoptosis. 5.3 Regulation of dexamethasone-induced apoptosis by PI3-kinase-Akt pathway (I) PI3-kinase-Akt-pathway is activated in a wide variety of cancers which results in enhanced resistance to apoptosis through multiple mechanisms. It has been shown that inhibition of PI3kinase decreases cell survival and enhances the effects of chemotherapeutic drugs in many types of

56 cancer cells (Asselin et al., 2001; Clark et al., 2002; Hu et al., 2002; Ng et al., 2000; Wang et al., 2002). We showed that Akt is constitutively phosphorylated in HF28RA cells (I, Fig.4C). Inhibition of PI3-kinase-Akt pathway by LY294002 and Akt inhibitor completely blocked the phosphorylation and enhanced considerably the sensitivity of HF28RA cells to dexamethasone (I, Fig.4). Interestingly, apoptosis proceeded with accelerated kinetics, indicating that active PI3kinase-Akt-pathway does not only protect from but also delays Dex-induced apoptosis (I, Fig.5). Dex-induced activation of caspase-3 and -8 was also enhanced and accelerated in the presence of LY294002 (Fig.10, I, Fig.6B). In spite of the importance of PI3-kinase-Akt as a survival-promoting pathway, treatment with inhibitors alone did not cause cell death, in contrast to the results obtained in some other cell models (Liu et al., 2001; Xu et al., 2003). 4h

8h

Dex 0.1µM - + - + - + - + LY 10 µM - - + + - - + +

16h

24h

- + - + - + - - + + - -

- + + + pro-caspase8 cleaved caspase8 pro-caspase3 cleaved caspase-3

Figure 10. PI3-kinase inhibitor, LY294002, enhances and accelerates Dex-induced activation of caspase-3 and -8. HF28RA cells were pre-incubated with 10 µM LY294002 for 1 h prior to the addition of 0.1 µM Dex for 4, 8, 16 and 24 h. After incubations, proteins were isolated and cleavage of caspase-3 and -8 was analyzed by Western blotting.

Furthermore, inhibition of PI3-kinase with LY294002 markedly enhanced Dex-induced apoptosis already at the mitochondrial level (I, Fig.6A, C). Previous findings support our results since it has been shown that Akt inhibited apoptosis at a pre-mitochondrial level inhibiting the cytochrome c release and the loss of mitochondrial membrane potential (Gottlob et al., 2001; Kennedy et al., 1999). One connection between Akt and mitochondria is the phosphorylation of the pro-apoptotic Bcl-2 family protein Bad. Bad is phosphorylated at Ser-136 by Akt and also at Ser-112 and at Ser155 by other kinases. Bad does not contain a mitochondrial targeting sequence but localizes to mitochondria, in a phosphorylation-dependent manner. When Bad is phosphorylated it binds to 143-3 proteins in the cytosol. Dephosphorylated Bad is released from 14-3-3 and becomes free to

57 heterodimerize with anti-apoptotic members of Bcl-2 family proteins (Bcl-2, Bcl-XL and Bcl-w) in mitochondria, thereby inhibiting their anti-apoptotic activity (Gross et al., 1999). Our results showed that inhibition of PI3-kinase induced translocation of Bad to mitochondria (I, Fig.7). Inhibition of PI3-kinase-Akt pathway did not alone induce apoptosis indicating that translocation of Bad to mitochondria itself is not enough to induce apoptosis but another apoptotic signal is required. It has been reported that Bim can bind not only to anti-apoptotic Bcl-2 family members but also to Bax and/or Bak (Huang and Strasser, 2000; Letai et al., 2002; Marani et al., 2002). In contrast, Bad has been reported to bind only Bcl-2 and its homologues but not to Bax or Bak (Letai et al., 2002). Accordingly, it has been postulated that Bim is a direct inducer of apoptosis, whereas Bad sensitizes cells to death stimuli by reducing the level of free anti-apoptotic Bcl-2 family proteins. To investigate the possible partners of Bad and Bim in mitochondria, the subcellular localization of Bax, Bcl-XL and Bcl-2 was analyzed. In agreement with previously published results, anti-apoptotic proteins Bcl-2 and Bcl-XL were predominantly present in mitochondria (I, Fig.7). The pro-apoptotic member of the Bcl-2 family, Bax, is thought to reside in the cytosol of healthy cells and in response to apoptotic stimuli, Bax undergoes a conformational change that leads to the translocation of Bax to mitochondria (Gross et al., 1998; Nechushtan et al., 1999). However, it has been also shown that Bax can be loosely associated with the outer mitochondrial membrane when not activated. Our results showed that Bax was predominantly present in mitochondria already in untreated HF28RA cells (I, Fig.7). In conclusion, based on our results and the current knowledge of Bim and Bad action, it seems that Bad is a sensitizer of apoptosis and other events induced by Dex, such as upregulation of Bim protein expression, are required to trigger apoptosis. It seems that translocation of Bad potentiates apoptosis by binding to antiapoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) freeing Bax and Bim to interact with each other and thereby leading to activation of Bax. Akt can also regulate cell survival by transcription-based mechanisms. Members of the forkhead family of transcription factors have been shown to be direct targets of Akt. Phosphorylation of forkhead proteins by Akt appears to alter their subcellular localization. In the absence of Akt activation, forkhead proteins are predominantly present in the nucleus where they are able to promote transcription of pro-apoptotic target genes (Nicholson and Anderson, 2002). It has been shown that PI3-kinase-Akt signaling inhibits Bim expression by phosphorylating the FOXO3 (forkhead box O3A; also known as FKHRL1) (Dijkers et al., 2002; Strasser, 2005). Therefore, we analyzed the effect of PI3-kinase inhibitor LY294002 on the expression of Bim. However,

58 inhibition of PI3-kinase had no effect on Bim expression in 24 h (I, Fig 7). In addition, further kinetic study showed similar results (Fig.11).

4h

8h

Dex 0.1µM - + - + - + - + LY 10 µM - - + + - - + +

16h

24h

- + - + - + - - + + - -

- + + + Bim EL Bim L Bim S

Figure 11. PI3-kinase inhibitor, LY294002, has no effect on Dex-induced upregulation of Bim. HF28RA cells were pre-incubated with 10 µM LY294002 for 1 h prior to the addition of 0.1 µM Dex for 4, 8, 16 and 24 h. After incubations, proteins were isolated and the amounts of Bim proteins were analyzed by Western blotting.

5.4 The role of glycogen synthase kinase-3 (GSK3) in Dex-induced apoptosis (II) GSK3 has been shown to promote the intrinsic apoptotic signaling induced by many different stimuli. GSK-3 facilitates the intrinsic apoptotic signaling through targeting several proteins that regulate signals leading to disruption of mitochondria (Beurel and Jope, 2006). Activity of GSK3 is negatively regulated through phosphorylation by Akt on Ser-21 in GSK3- or Ser-9 in GSK3- . However, several other kinases have also been shown to phosphorylate these inhibitory sites. The involvement of GSK3 in Dex-induced apoptosis was examined by using two structurally different GSK3 inhibitors, LiCl and SB-216763. Although both of the inhibitors inhibit also other targets, the only common target is GSK3 (Cohen and Goedert, 2004). To confirm that GSK is inhibited with LiCl and SB-216763, the inhibitory phosphorylation status of both isoforms of GSK3 was studied. Interestingly, the inhibitory phosphorylations by these inhibitors were dependent on subcellular localization of GSK3 / since the amount of phosphorylated GSK3 and GSK3 (at inhibitory site) increased in cytosol in SB-216763- treated cells and in mitochondria in LiCl-treated cells (II, Fig.2D). It has been previously shown that lithium increases the Ser-9 phosphorylation of GSK3 (Zhang et al., 2003). It has been also shown that lithium induces activation of Akt (Tajes et al., 2009). In addition, following PI3-kinase activation, Akt was shown to translocate to the mitochondria and phosphorylate Ser-9 of mitochondrial GSK3 (Bijur and Jope, 2003) Therefore, one mechanism how LiCl might increase inhibitory phosphorylations of GSK3 / at mitochondria could be that LiCl activates Akt which subsequently translocates to mitochondria and

59 phosphorylates mitochondrial GSK3 / at inhibitory serine residues. In contrast to our results, SB216763 which acts as an ATP competitor has been shown to inhibit GSK3 but not cause increase in Ser21/9 phosphorylation (Zhang, 2003). Furthermore, we showed that both inhibitors of GSK3 attenuated Dex-induced up-regulation of Bim, loss of mitochondrial membrane potential, release of cyt c and DNA fragmentation (II, Fig.2). These results indicate that GSK-3 contributes to Dex-induced apoptosis by regulating the expression of Bim. It has also been shown that GSK3 is required for trophic deprivation-induced expression of Bim in neurons (Hongisto et al., 2003). It has also been shown in osteoblast cells, that glucocorticoids induce apoptosis through the activation of GSK3 (Yun et al., 2009). However, to date, there are no previous studies demonstrating the involvement of GSK3 in the Dex-induced upregulation of Bim. FOXO transcription factors regulate the expression of Bim (as discussed earlier). However, it has been shown that Bim induction requires simultaneous activation of three different death signaling pathways (FOXO, Mybs, c-Jun) in neurons (Biswas et al., 2007). In future we will study whether FOXO transcription factors are responsible for Dex-induced upregulation of Bim, and how GSK3 contributes to Bim regulation. It would be of great interest also to see whether PI3-kinase-Akt pathway controls GSK3 activity in HF28RA cells. In contrast to our results, it has been shown that GSK3 has also inhibitory effect on glucocorticoidinduced apoptosis. It has been recently shown that GSK-3 -mediated GR phosphorylation inhibited glucocorticoid-dependent NF-kappaB transrepression and attenuated the glucocorticoid-dependent cell death of osteoblasts (Galliher-Beckley et al., 2008). Additionally, it has been shown that GSK3-inhibitor (TDZD) induced apoptosis in myeloma cell lines and TDZD-mediated inhibition of GSK3 resulted in dephosphorylation and activation of FOXO3a (Zhou et al., 2008). 5.5 Molecular mechanisms of TRAIL-induced apoptosis (III) TRAIL induces apoptosis through engaging TRAIL-R4 (DR4) and/or TRAIL-R5 (DR5). Both receptors are expressed normally simultaneously on the same cell. To investigate the expression of TRAIL-R1 and TRAIL-R2 on HF28RA and HF1A3 cell lines, cells were stained with Abs against the receptors. FACS analysis showed that both cell lines expressed both receptors on the cell surface (Fig.12A, B). In spite of the fact that both receptors are expressed on the same cell, generally TRAIL-R2 seems to play a more important role than TRAIL-R1 in triggering apoptosis (Kelley et al., 2005).

60 Antibody inhibition study was done to resolve, whether both TRAIL receptors mediate apoptosis in HF28RA and HF1A3 cell lines. Both blocking Abs (TRAIL-R1 or TRAIL-R2 mAb) decreased TRAIL-induced apoptosis indicating that both of the receptors were capable of mediating apoptosis in HF28RA and HF1A3 cells. Simultaneous TRAIL-R1 and TRAIL-R2 blockade almost completely prevented TRAIL-induced apoptosis in both cell lines (Fig.12C).

A)

B) HF1A3

Anti-mouse PE TRAIL-R1+ anti-mouse PE TRAIL-R2+ anti-mouse PE

PE

Counts

Counts

HF28RA

Anti-mouse PE TRAIL-R1+ anti-mouse PE TRAIL-R2+ anti-mouse PE

PE

Figure 12. Functional expression of TRAIL-receptors on HF28RA and HF1A3 cells. A-B) HF28RA and HF1A3 cells were stained with mAbs against TRAIL-R1 and TRAIL-R2 followed by PE-labeled goat anti-mouse. Isotype matched mouse IgG1 was used as control. Stained cells were analyzed by flow cytometry and histograms of the FACS analysis are presented. C) Cells were incubated with TRAIL-R1 and/or TRAIL-R2 mAb for 30 min to block either one or both of the receptors, prior to addition of TRAIL. After 24 h, the percentage of apoptotic cells was analyzed by propidium iodide staining. The data are presented as a mean + SEM from three independent experiments.

61

To study whether caspase-8 is required for TRAIL-induced apoptosis, cells were pre-incubated with 15 µM caspase-8 inhibitor, Z-IETD-FMK for 1 hour prior addition of TRAIL for 24 h. As expected, Z-IETD-FMK completely prevented TRAIL-induced apoptosis in both cell lines (Fig. 13).

Figure 13. Inhibition of caspase-8 prevents TRAIL-induced apoptosis. Cells were pre-incubated with 15 µM caspase-8 inhibitor, Z-IETD-FMK for 1 hour prior to addition of TRAIL for 24 h. The percentage of apoptotic cells was analyzed by propidium iodide staining. The data are presented as a mean + SEM from three independent experiments.

Besides caspase-8, caspase-10 can also be recruited to the TRAIL DISC (Kischkel et al., 2001). However, the importance of caspase-10 in induction of apoptosis is controversial (Kischkel et al., 2001; Sprick et al., 2002). To investigate the potential role of caspase-10 during TRAIL-induced apoptosis, cells were transduced with dominant negative (DN) form of caspase-10 in IRES-GFPlentivector. Overexpression of DN-caspase-10 was confirmed by Western blotting (Fig. 14A). HF28RA and HF1A3 cells transduced with "empty" IRES-GFP lentivirus vector were used as controls (HF28RA GFP, HF1A3 GFP). In HF1A3 cells, caspase-10 is not involved in TRAILinduced apoptosis as the DN form of caspase-10 had no effect on TRAIL-induced apoptosis (Fig.14B). In contrast, in HF28RA cells, DN caspase-10 decreased TRAIL-induced apoptosis indicating that caspase-10 may play a minor role (Fig.14B).

62 A)

HF1A3

HF28RA

DNDNGFP casp10 GFP casp10

Figure 14. The role of caspase-10 in TRAIL-induced apoptosis. A) Total cellular proteins were isolated from vector control (GFP expressing) cells and dominant- negative caspase-10 expressing cells and western blot analysis was performed to confirm the overexpression of DN-caspase-10. B) Cells were treated with 50 ng/ml TRAIL for 24 h and percentage of apoptotic cells was analyzed by propidium iodide staining. The data are presented as a mean + SEM from three independent experiments. 5.5.1 Type I and type II TRAIL signaling (III) Cells can be divided into two types according to their requirement for mitochondria in death receptor induced apoptosis. It has been suggested that in type I cells apoptosis can proceed independently of mitochondria. In type II cells, apoptosis is thought to depend on mitochondria and is blocked by overexpression of Bcl-2 or Bcl-XL (Scaffidi et al., 1998). In TRAIL-signaling both apoptotic pathways have also been described. It has been shown that overexpression of Bcl-2 or Bcl-XL does not block TRAIL-induced apoptosis in some cancer cell lines (Fulda et al., 2002; Keogh et al., 2000; Kim et al., 2001; Rudner et al., 2001; Walczak et al., 2000). In contrast, overexpression of Bcl-2 or Bcl-XL blocks or reduces TRAIL-induced apoptosis in other cancer cell lines (Fulda et al., 2002; Hinz et al., 2000; Munshi et al., 2001; Rokhlin et al., 2001; Ruiz de Almodovar et al., 2001; Srinivasula et al., 2000; Sun et al., 2001). We showed that mitochondria have an important but distinct role during TRAIL-induced apoptosis in FL cell lines. Mitochondrial changes (loss of

m and release of cyt c) were faster and more strongly activated in HF28RA

63 cells than in HF1A3 cells while DNA fragmentation followed similar quantity and kinetics (III, Fig.1). In addition, Bcl-XL overexpression studies revealed that in HF28RA cells TRAIL-induced apoptosis is completely dependent on the mitochondrial activation (III, Fig.1). Recently the requirement of mitochondrial pathway in type II cells was challenged as it was shown that protective effect of Bcl-2 was limited to lower concentrations of TRAIL or early observation time points (Rudner et al., 2005). To study whether increasing amount of TRAIL or duration of TRAIL stimulus, could affect the mitochondrial dependence, cells were incubated with 50 ng/ml or 100 ng/ml TRAIL for 24 and 48 hours. The percentage of cells with depolarized mitochondrial membrane and the percentage of apoptotic cells were measured by TMRM- and PI-stainings, respectively. The results show that higher concentration of TRAIL or prolonged incubation time did not change type II signaling to type I signaling, as the Bcl-XL still prevented all TRAILinduced apoptotic changes in HF28RA-cells (Fig.15A, B).

Figure 15. Increasing concentration of TRAIL and duration of TRAIL stimulus has no effect on type II behavior. Cells were incubated with 50 ng/ml or 100 ng/ml TRAIL for 24 or 48 hours and (A) percentage of apoptotic cells was analyzed by propidium iodide staining and (B) the percentage of cells with depolarized mitochondrial membrane was analyzed by TMRM staining.

64 In contrast, in HF1A3 cells, although overexpression of Bcl-XL almost completely blocked depolarization of mitochondrial membrane, it only decreased DNA fragmentation (III, Fig.1E, G). Interestingly, in Bcl-XL overexpressing HF1A3 cells TRAIL-induced apoptosis was decreased much lesser extent in 4 h than at later time points (8h and 16h) (III, Fig.1) indicating that mitochondria might serve a late amplification loop to caspase activation in HF1A3 cells. In conclusion, due to these differences in mitochondrial involvement during TRAIL-induced apoptosis, we suggest that HF28RA cells represent type II cells and HF1A3 cells resemble more type I cells. These differences in mitochondrial activation could not be explained by different amount of Bcl-2 or Bcl-XL proteins because it has been previously shown that these cell lines express comparable levels of Bcl-2 (Skommer et al., 2006) and we showed that amount of Bcl-XL was even slightly higher in HF1A3 cells than in HF28RA cells (III, Fig.1A). Interestingly, overexpression of Bcl-XL failed to prevent TRAIL-induced release of mitochondrial cyt c to the cytosol in HF1A3 cells even the loss of mitochondrial membrane potential was prevented (III, Fig.1E, F) indicating that these are independent events. In contrast, release of cyt c was associated with the loss of

m

in HF28RA cells. Generally, the release of apoptotic proteins

from mitochondria, such as cyt c, is thought to be associated with the loss of

m.

However, it has

been shown that release of cyt c is independent of mitochondrial depolarization (Bossy-Wetzel et al., 1998) and that

m can still be maintained after the release of cyt c to cytosol (Waterhouse et

al., 2001). It was recently shown that also in death receptor induced (Fas and TRAIL) apoptosis depolarization of mitochondrial membrane potential can be uncoupled from cyt c release (Samraj et al., 2006). It is known that mitochondria are activated in both cell types, however it is not yet understood why type II cells exclusively utilize mitochondrial pathway in death receptor mediated apoptosis. It has been suggested that the amount of activated caspase-8 generated at the DISC may be one critical factor (Scaffidi et al., 1998). Our results show that large amount of caspase-8 was activated in both cell lines after TRAIL treatment (III, Fig.2). It has been previously shown that overexpression of Bcl-XL or Bcl-2 blocks activation of caspase-8 in type II cells but not in type I cells (Scaffidi et al., 1998). In addition, in Jurkat cells (type II) overexpression of Bcl-XL inhibited Fas-induced apoptosis and also caspase-8 activation (Zhao et al., 2007). Our data showed that Bcl-XL did not prevent or even reduce the activation of caspase-8 in either of cell types (III, Fig.2). In addition, Bid was cleaved equally in both cell lines and overexpression of Bcl-XL did not prevent cleavage of Bid (III, Fig.2). Therefore, it seems unlikely that neither the amount of caspase-8 activation at the DISC nor the Bid cleavage can define whether cells are type I or type II cells, at least in our model.

65 On the contrary to our result, it has been shown in glioma cells that enforced overexpression of caspase-8 in type II cells overcomes Bcl-2 inhibition of Fas- and TRAIL-induced apoptosis, converting cells from type II to type I (Knight et al., 2004). In addition, it was shown that hepatocytes (type II cells) deficient in Bid were resistant to Fas-induced apoptosis and high dose of hepatocyte growth factor (HGF) sensitized Bid deficient cells to Fas indicating a decrease in mitochondria requirement (Zhao et al., 2007). It is commonly thought that release of cyt c leads to the formation of apoptosome and activation of caspase-9. However, there are not much data about the role of caspase-9 in TRAIL-induced apoptosis. In type II human hepatocytes and colon carcinoma cells it has been previously shown that caspase-9 inhibitor completely blocked TRAIL-induced apoptosis (Ozoren et al., 2000). In contrast, in colon cancer cells caspase-9 inhibition failed to block TRAIL-induced apoptosis (Ozoren et al., 2000). We show that overexpression of DN caspase-9 had no effect on TRAILinduced apoptosis in HF1A3 cells (III, Fig.3E-G) despite the fact that cyt c was released. In contrast, in HF28RA cells DN caspase-9 decreased TRAIL-induced DNA fragmentation, but surprisingly also mitochondrial changes (III, Fig.3B-D), even though caspase-9 is thought to locate downstream from mitochondria in the apoptotic cascade. These results indicate that there might be a late amplification loop to mitochondria through caspase-9. Interestingly, DN-caspase-9 failed to completely prevent TRAIL-induced apoptosis in HF28RA cells (III, Fig.3B-D) indicating that other factors, besides cyt c, could be released from mitochondria. Indeed, the activation of mitochondrial apoptotic activity may also result in the release of several other pro-apoptotic factors, such as Smac/DIABLO, HtrA2/Omi, AIF and EndoG. The sensitivity of HF28RA cells to TRAIL apoptosis in spite of overexpression of DN caspase-9 is, therefore, not surprising. It has been previously shown that TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO (Deng et al., 2002). We could not detect the release of mitochondrial Smac/DIABLO during TRAIL-induced apoptosis in HF28RA and HF1A3 cells (data not shown). Nevertheless, it seems likely that in type II cells, release of a pro-apoptotic protein(s) from mitochondria, other than cyt c or Smac/DIABLO, is/are required before caspases can be fully activated.

66 Table 1. Effect of Bcl-XL or DN-caspase-9 overexpression on TRAIL-induced apoptotic changes in HF28RA and HF1A3 cells. Effect of overexpression of Bcl-XL on caspase-8 activation cleavage of Bid loss of m release of cyt c apoptosis

HF28RA (type II) -/+ -/+ -------

Effect of overexpression of DN-caspase9 on loss of m - (in 8h) release of cyt c -apoptosis +/- no effect, - slight decrease, -- decrease, --- prevention

HF1A3 (type I-like) -/+ -/+ --- (in 4h) -- (in 8h)

-/+ -/+ -/+

Furthermore, we showed that PDTC, a potent inhibitor of NF- B, enhanced TRAIL-induced caspase-8 activation, depolarization of mitochondrial membrane and DNA fragmentation in HF28RA cells but had no effect on HF1A3 cells (III, Fig.4). Interestingly, in HF28RA cells overexpression of Bcl-XL failed to prevent TRAIL-induced DNA fragmentation in the presence of PDTC even the mitochondrial membrane depolarization (III, Fig.4A, B) and release of cyt c (Fig.16) were still blocked. Dominant negative FADD prevented TRAIL-induced loss of mitochondrial membrane potential and apoptosis in the absence and presence of PDTC, proving that the death signal was still originated from the TRAIL receptor when PDTC and TRAIL were used in combination (III, Fig.4E, F). Thus, our data show that PDTC switches TRAIL-induced apoptotic pathway from mitochondrial dependent type II to mitochondria-independent type I pathway in HF28RA cells. However, we showed that another more specific inhibitor of NF- B pathway, IKK inhibitor, enhanced TRAIL-induced apoptosis in both cell types and did not switch apoptosis to type I pathway in Bcl-XL overexpressing type II cells (III, Fig.5) indicating that NF- B might not be the target of PDTC being responsible for the switch. Therefore, future studies are required to solve the target of PDTC that regulates the requirement of mitochondria in TRAILinduced apoptosis. In pancreatic carcinoma cells, it was shown that XIAP knockdown by RNA interference converted Bcl-2 overexpressing type II cells to type I cells in response to TRAIL (Vogler et al., 2008). It was also shown in childhood acute leukemia cells that small molecule XIAP inhibitors enhance TRAIL-

67 induced apoptotic changes and cooperate with TRAIL to overcome Bcl-2-mediated resistance (Fakler et al., 2009). However, in both of these studies inhibition of XIAP in combination with TRAIL induced loss of

m

and release of cyt c in Bcl-2 overexpressing cells (Fakler et al., 2009;

Vogler et al., 2008). Furthermore, it has been shown that sublethal dose of protein synthesis inhibitor, cycloheximide (CHX), overcomes Bcl-2 protection in type II cells in response to Fas. However, CHX induced the loss of mitochondrial membrane potential and release of cyt c in combination with anti-Fas (Brumatti et al., 2008). It has also been shown in TRAIL-resistant colon carcinoma cells, proteosome inhibitors sensitizied cells to TRAIL (Nagy et al., 2006). In that study, TRAIL alone induced partial caspase-3 activation, while the combination of TRAIL and proteosome inhibition led to the full proteolytic activation of caspase-3. Enhanced release of mitochondrial Smac/DIABLO to the cytosol was also shown indicating that the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO (Nagy et al., 2006). Several other studies have also shown that proteosome inhibitors sensitize TRAIL resistant cells to TRAIL and induce full activation of caspase-3 (Leverkus et al., 2003; Zhu et al., 2005). We also studied the activation of caspase-3 by Western blotting using an antibody that recognizes the cleavage products of caspase-3. In HF28RA GFP cells (vector control cells), TRAIL induced full activation of caspase-3 which was further enhanced by PDTC. Interestingly, in Bcl-XL overexpressing cells, TRAIL induced only partial activation of caspase-3 and the presence of PDTC led to the full activation of caspase-3 (Fig.16).

HF28RA GFP HF28RA Bcl-XL Trail Trail PDTC + PDTC + Co 0.2 µM Trail PDTC Co 0.2 µM Trail PDTC cyt c cleaved caspase-3

Figure 16. Effect of PDTC on TRAIL-induced release of cytochrome c and cleavage of caspase-3 in HF28RA GFP and HF28RA Bcl-XL cells. Cells were treated with 0.2 µM PDTC for 2h prior to addition of 50 ng/ml TRAIL for additional 4h. After stimulations, cytosolic and mitochondrial proteins were separated and the amounts of cytosolic cyt c and cleavage products of caspase-3 were analyzed by Western blotting.

68

One hypothesis, based on the results from caspase-3 activation (Fig.16), is that Bcl-XL prevents the release of mitochondrial protein x which is normally released to cytosol after TRAIL treatment and required for full activation of caspase-3 probably by releasing partially processed caspase-3 from inhibition. Furthermore, PDTC might directly or indirectly inhibit the inhibitor enabling the full activation of caspase-3 without mitochondrial contribution (Fig.17).

Figure 17. Hypothesis of how PDTC enables type I signaling in BCL-XL overexpressing type II cells.

5.6 The effect of microenvironmental CD40 signaling on dexamethasone-, doxorubicin- and TRAIL-induced apoptosis (IV) Despite many different treatment approaches, the overall survival time of patients with follicular lymphoma has not improved and it is still considered to be incurable. The progression of FL varies between the patients, as about 15% of FL patients have a poor prognosis due to transformation to aggressive disease and die within 3 years of diagnosis, but 20-25% may live more than 15 years after diagnosis (Dave et al., 2004; de Jong, 2005). Recent studies have shown that the cellular

69 microenvironment influences the progression of FL (Alvaro et al., 2006; Dave et al., 2004; Glas et al., 2005). FL cells are malignant counterparts of germinal center (GC) B cells, so like normal GC B cells, FL cells interact with various immune cells, such as follicular helper T cells and dendritic cells that define the tumor microenvironment (Martinez et al., 2008). CD40 ligation (CD40 receptor on GC B cells and CD40L on helper T cells) is known to be an important mediator of survival signals in GCs and might have an impact on treatment of follicular lymphoma. It has been shown that CD40 stimulation rescues FL B cells from Fas-, B cell receptor (BCR)-, rituximab- and TRAIL-induced apoptosis (Eeva et al., 2003; Eeva et al., 2007; Eeva et al., 2009; Travert et al., 2008). We tested responses of CD40 signaling on dexamethasone- and doxorubicin-induced apoptosis, both of which are currently used in treatment protocols of FL, as well as on TRAIL-induced apoptosis which is in clinical trials, in three human follicular lymphoma cell lines. The cell lines have previously been characterized according to surface marker expression and it was shown that follicular lymphoma HF4.9 cells originate from the earlier maturation stage of germinal center development than HF28RA and HF1A3 cells (Eray et al., 2003). Our results showed that CD40 stimulation protected HF28RA and HF1A3 cells from dexamethasone-, doxorubicin- and TRAIL-induced apoptosis (IV, Fig.1). On the contrary, CD40 itself induced apoptosis in HF4.9 cells (IV, Fig.5). This result raised a question whether maturation stage of FL cells determines the outcome of CD40 signaling. In lymphoma B cells both CD40-induced proliferation/survival and induction of growth arrest/apoptosis have been reported (Dallman et al., 2003). CD40 ligation has been shown to decrease the proliferation or induce apoptosis in high grade B cell lymphomas (Funakoshi et al., 1994; Wang et al., 2008). In contrast, in low-grade B cell malignancies CD40 has been shown to promote survival (Ghia et al., 1998; Travert et al., 2008). CD40 has been shown to activate multiple signaling pathways. However, signaling pathways responsible for anti-apoptotic function are not fully understood. Many studies have shown that CD40 induces activation of NF- B (Berberich et al., 1994; Coope et al., 2002; Homig-Holzel et al., 2008; Lee et al., 1999; Travert et al., 2008). In B cells, NF- B is constitutively active but can be further induced (Liu et al., 1991). Our EMSA results also showed that NF- B was constitutively active but the activation could be strongly enhanced by CD40 (IV, Fig. 2B). Recently it was shown that CD40 stimulation protects FL cells from TRAIL-induced apoptosis through activation of NFB (Travert et al., 2008). We used two different NF- B pathway inhibitors, antioxidant pyrrolidine dithiocarbamate (PDTC) and specific I B kinase (IKK)-inhibitor (BMS-345541) to study the role

70 of NF- B in CD40 protection. PDTC has been shown to inhibit NF- B activation (Wang et al., 2008) and our results also show that PDTC prevented completely CD40-induced activation of NFB (IV, Fig.2B). Specific IKK-inhibitor has been shown to inhibit both IKK and IKK (Burke et al., 2003). In two of the FL cell lines in which CD40 stimulation protected from TRAIL- and cytotoxic drug-induced apoptosis, inhibition of NF- B prevented the protection indicating that CD40 induced protection is completely dependent on NF- B (IV, Fig.2A, 3A). This was shown only with TRAIL-induced apoptosis which is induced already in 4 h because inhibition of NF- B pathway became toxic itself at the later time points as the number of apoptotic cells started to increase slowly after four hours (data not shown). In addition, we show that CD40 could not protect from NF- B-inhibitor induced apoptosis confirming that CD40 signaling that leads to protection from apoptosis requires NF- B (IV, Fig.3B). In contrast, it was recently shown that CD40 ligation mediated inhibition of NF- B and promoted apoptosis in human Burkitt lymphoma cell lines (Wang et al., 2008). In HF4.9 cells, CD40 might also induce apoptosis by inhibition of NF- B but further studies are still required to prove that hypothesis. Inhibition of NF- B induced apoptosis in all three FL cell lines (IV, Fig.3B, 5B) indicating that there is basal level of NF- B-regulated anti-apoptotic proteins in a cell which is not enough to prevent TRAIL- or cytotoxic drug-induced apoptosis but is enough to protect from spontaneous apoptosis. Furthermore, our results showed that inhibition of NF- B further sensitized cells to apoptosis (IV, Fig.2A, 3A). It has also been previously shown that inhibition of IKK sensitizes mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level (Roue et al., 2007). In addition, it has been shown that CD40 induces up-regulation of c-FLIP and Bcl-XL and that the induction is partially prevented by selective NF- B inhibitor, BAY 117085 (Travert et al., 2008). Our RT-PCR results showed that CD40 induced expression of IAP family of proteins (c-IAP1, cIAP-2, XIAP, survivin), Bcl-XL and c-FLIPs (c-FLIP-L, c-FLIP-S) and the induction was completely prevented by NF- B inhibition (IV, Fig.2C, 3C). At the protein level, CD40 induction of these anti-apoptotic proteins was much less prominent. However, inhibition of NF- B decreased considerably amounts of Bcl-XL and FLIP-S/L proteins (basal level) (IV, Fig.3D). The anti-apoptotic proteins, FLIP-S and FLIP-L, block death receptor induced apoptosis inhibiting activation of caspase-8 at the death inducing signaling complex (DISC) (Krueger et al., 2001; Scaffidi et al., 1999). Overexpression of either of the FLIP isoforms inhibited TRAIL-induced apoptosis but as supposed not intrinsic apoptosis induced by dexamethasone or doxorubicin (IV, Fig.4). Follicular lymphomas overexpress Bcl-2 protein but it is not itself enough to prevent

71 TRAIL-, doxorubicin- or dexamethasone-induced apoptosis. Here we show that simultaneous overexpression of Bcl-XL prevented almost completely dexamethasone- and doxorubicin-induced apoptosis (IV, Fig.4B). In addition, Bcl-XL prevented TRAIL-induced apoptosis in mitochondria dependent type II FL cells (HF28RA) but as supposed inhibited only partially TRAIL-induced apoptosis in mitochondria independent type I FL cells (HF1A3) (discussed previously, III, Fig.1 ).

72 6. CONCLUSIONS Glucocorticoids such as dexamethasone are frequently used in the treatment of lymphoid malignancies due to their ability to induce apoptosis in lymphoid cells. However, the detailed mechanisms and regulation of Dex-induced apoptosis remain still largely unknown in FL cells. Dex-induced apoptosis depends on mitochondria since overexpression of Bcl-XL completely prevented Dex-induced apoptosis. Protein synthesis is required for Dex-induced apoptosis already before mitochondrial changes. The kinetics of Dex-induced upregulation of Bim correlates with kinetics of apoptosis indicating that newly synthetized Bim might be the central mediator of Dexinduced apoptosis. However, sh-RNA knockdown of Bim is required to prove the result. Interestingly, activity of GSK-3 is required for Dex-induced upregulation of Bim and apoptosis. Surprisingly, caspase-9 which is normally thought to be activated downstream of mitochondria, regulates Dex-induced mitochondrial changes since overexpression of dominant negative caspase-9 prevented Dex-induced mitochondrial changes and apoptosis. (Fig. 18)

Figure 18. Mechanisms and regulation of Dex-induced apoptosis.

73 The inhibition of constitutively activated PI3-kinase-Akt pathway in FL cells enhances and accelerates Dex-induced apoptosis indicating the importance of this pathway in the protection of follicular lymphoma cells from cell death. Therefore modulation of PI3-kinase-Akt pathway activity could provide new strategies to improve current therapeutic regimens in FL. Specific inhibitors of PI3-kinase-Akt pathway should be considered in the future in the treatments of lymphoid malignancies in combination with glucocorticoids. In addition, shifting the balance of the activation of the kinases towards the state where glycogen synthase kinase-3 is kept active favors an apoptotic response and could potentiate Dex-induced apoptosis. TRAIL has gained lately a lot of attention because of its ability to induce apoptosis in cancer cells leaving the normal cells intact. TRAIL induces apoptosis either by type I or type II pathway in FL cells (Fig.19). Interestingly, in the presence of PDTC, TRAIL induced the full activation of caspase-3 and apoptosis independently of mitochondria in Bcl-XL overexpressing type II cells indicating that apoptotic pathway switches from type II to type I. However, the target of PDTC remains to be solved. The activation of apoptosis independently of mitochondria could have great advantage to treatment of FL because most cases of FL overexpress anti-apoptotic mitochondrial Bcl-2 protein which either protects or delays apoptosis.

Figure 19. The possible mechanisms of type I and type II apoptotic pathways in FL cells in response to TRAIL.

74 Furthermore, our results suggest that microenvironmental CD40 signals might have an impact on treatment of FL. Interestingly, CD40 stimulation either protected from TRAIL- and drug-induced apoptosis or induced apoptosis itself, depending on the maturation stage of FL B cells. Therefore, the use of blocking CD40 antibodies in the treatment might not bring advantage for all FL patients. Instead, since CD40 protection was dependent on NF- B and inhibition of NF- B induced apoptosis in all three FL cell lines, the best combination for FL therapy according to our in vitro studies could be pharmacological inhibitors of NF- B combined with TRAIL or other cytotoxic drugs. Although inhibitors of NF-

increasingly are being used for treatment of human

malignancies, serious side effects limit their clinical use. Thus, a preferable approach would be to block the critical downstream anti-apoptotic targets of NF-

rather than NF-

itself.

75 7. REFERENCES

Abrams MT, Robertson NM, Yoon K, Wickstrom E. Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA. J.Biol.Chem. 2004;279:55809-55817. Adams JM, Cory S. Life-or-death decisions by the bcl-2 protein family. Trends Biochem.Sci. 2001;26:61-66. Alexanian R, Dimopoulos M. The treatment of multiple myeloma. N.Engl.J.Med. 1994;330:484489. Alnemri ES, Fernandes TF, Haldar S, Croce CM, Litwack G. Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B-leukemias. Cancer Res. 1992;52:491-495. Alvaro T, Lejeune M, Salvado MT, Lopez C, Jaen J, Bosch R et al. Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients. J.Clin.Oncol. 2006;24:5350-5357. Asselin E, Mills GB, Tsang BK. XIAP regulates akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells. Cancer Res. 2001;61:1862-1868. Ausserlechner MJ, Obexer P, Bock G, Geley S, Kofler R. Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells. Cell Death Differ. 2004;11:165-174. Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL, Cooke MP. Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol.Cell 2003;12:627-637. Bachmann PS, Gorman R, Mackenzie KL, Lutze-Mann L, Lock RB. Dexamethasone resistance in B-cell precursor childhood acute lymphoblastic leukemia occurs downstream of ligand-induced nuclear translocation of the glucocorticoid receptor. Blood 2005;105:2519-2526. Bendandi M. Aiming at a curative strategy for follicular lymphoma. CA Cancer.J.Clin. 2008;58:305-317. Berberich I, Shu GL, Clark EA. Cross-linking CD40 on B cells rapidly activates nuclear factorkappa B. J.Immunol. 1994;153:4357-4366. Beurel E, Jope RS. The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog.Neurobiol. 2006;79:173-189. Beurel E, Kornprobst M, Blivet-Van Eggelpoel MJ, Cadoret A, Capeau J, Desbois-Mouthon C. GSK-3beta reactivation with LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis. Int.J.Oncol. 2005;27:215-222.

76 Beurel E, Kornprobst M, Blivet-Van Eggelpoel MJ, Ruiz-Ruiz C, Cadoret A, Capeau J et al. GSK3beta inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression. Exp.Cell Res. 2004;300:354-364. Bijur GN, De Sarno P, Jope RS. Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. protection by lithium. J.Biol.Chem. 2000;275:7583-7590. Bijur GN, Jope RS. Glycogen synthase kinase-3 beta is highly activated in nuclei and mitochondria. Neuroreport 2003;14:2415-2419. Bijur GN, Jope RS. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3kinase activation. J. Neurochem. 2003;87:1427-1435. Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW. Bcl-XL inhibits membrane permeabilization by competing with bax. PLoS Biol. 2008;6:e147. Biswas SC, Shi Y, Sproul A, Greene LA. Pro-apoptotic bim induction in response to nerve growth factor deprivation requires simultaneous activation of three different death signaling pathways. J.Biol.Chem. 2007;282:29368-29374. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat.Cell Biol. 2000;2:241-243. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 1998;17:37-49. Bouillet P, Strasser A. BH3-only proteins - evolutionarily conserved proapoptotic bcl-2 family members essential for initiating programmed cell death. J.Cell.Sci. 2002;115:1567-1574. Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001;61:3986-3997. Brumatti G, Yon M, Castro FA, Bueno-da-Silva AE, Jacysyn JF, Brunner T et al. Conversion of CD95 (fas) type II into type I signaling by sub-lethal doses of cycloheximide. Exp.Cell Res. 2008;314:554-563. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 1999;96:857-868. Bullock BP, Habener JF. Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge. Biochemistry 1998;37:3795-3809. Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J.Biol.Chem. 2003;278:1450-1456.

77 Camilleri-Broet S, Vanderwerff H, Caldwell E, Hockenbery D. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp.Cell Res. 1998;239:277-292. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318-1321. Caron-Leslie LA, Evans RB, Cidlowski JA. Bcl-2 inhibits glucocorticoid-induced apoptosis but only partially blocks calcium ionophore or cycloheximide-regulated apoptosis in S49 cells. FASEB J. 1994;8:639-645. Cartron PF, Gallenne T, Bougras G, Gautier F, Manero F, Vusio P et al. The first alpha helix of bax plays a necessary role in its ligand-induced activation by the BH3-only proteins bid and PUMA. Mol.Cell 2004;16:807-818. Casale F, Addeo R, D'Angelo V, Indolfi P, Poggi V, Morgera C et al. Determination of the in vivo effects of prednisone on bcl-2 family protein expression in childhood acute lymphoblastic leukemia. Int.J.Oncol. 2003;22:123-128. Castedo M, Macho A, Zamzami N, Hirsch T, Marchetti P, Uriel J et al. Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo. Eur.J.Immunol. 1995;25:3277-3284. Cataldi A, Zauli G, Di Pietro R, Castorina S, Rana R. Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation. Cell.Signal. 2001;13:369-375. Certo M, Del Gaizo Moore V, Nishino M, Wei G, Korsmeyer S, Armstrong SA et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer.Cell. 2006;9:351-365. Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NFkappaB pathway. Immunity 1997;7:821-830. Chauhan D, Auclair D, Robinson EK, Hideshima T, Li G, Podar K et al. Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays. Oncogene 2002;21:1346-1358. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG et al. Differential targeting of prosurvival bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol.Cell 2005;17:393-403. Cheng EH, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 2003;301:513-517. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol.Cell 2001;8:705-711.

78 Chipuk JE, Bouchier-Hayes L, Green DR. Mitochondrial outer membrane permeabilization during apoptosis: The innocent bystander scenario. Cell Death Differ. 2006;13:1396-1402. Cifone MG, Migliorati G, Parroni R, Marchetti C, Millimaggi D, Santoni A et al. Dexamethasoneinduced thymocyte apoptosis: Apoptotic signal involves the sequential activation of phosphoinositide-specific phospholipase C, acidic sphingomyelinase, and caspases. Blood 1999;93:2282-2296. Clancy L, Mruk K, Archer K, Woelfel M, Mongkolsapaya J, Screaton G et al. Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis. Proc.Natl.Acad.Sci.U.S.A. 2005;102:18099-18104. Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol.Cancer.Ther. 2002;1:707-717. Cohen P, Goedert M. GSK3 inhibitors: Development and therapeutic potential. Nat.Rev.Drug Discov. 2004;3:479-487. Coope HJ, Atkinson PG, Huhse B, Belich M, Janzen J, Holman MJ et al. CD40 regulates the processing of NF-kappaB2 p100 to p52. EMBO J. 2002;21:5375-5385. Cretney E, Takeda K, Yagita H, Glaccum M, Peschon JJ, Smyth MJ. Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice. J.Immunol. 2002;168:1356-1361. Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F. X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res. 2004;64:30063008. Dallman C, Johnson PW, Packham G. Differential regulation of cell survival by CD40. Apoptosis 2003;8:45-53. Dan HC, Sun M, Kaneko S, Feldman RI, Nicosia SV, Wang HG et al. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J.Biol.Chem. 2004;279:5405-5412. Dave SS, Wright G, Tan B, Rosenwald A, Gascoyne RD, Chan WC et al. Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N.Engl.J.Med. 2004;351:2159-2169. de Jong D. Molecular pathogenesis of follicular lymphoma: A cross talk of genetic and immunologic factors. J.Clin.Oncol. 2005;23:6358-6363. Deng Y, Lin Y, Wu X. TRAIL-induced apoptosis requires bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev. 2002;16:33-45. Diehl GE, Yue HH, Hsieh K, Kuang AA, Ho M, Morici LA et al. TRAIL-R as a negative regulator of innate immune cell responses. Immunity 2004;21:877-889.

79 Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Koenderman L et al. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: Protein kinase Benhanced cell survival through maintenance of mitochondrial integrity. J.Cell Biol. 2002;156:531542. Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ. Expression of the pro-apoptotic bcl-2 family member bim is regulated by the forkhead transcription factor FKHR-L1. Curr.Biol. 2000;10:1201-1204. DiSanto JP, Bonnefoy JY, Gauchat JF, Fischer A, de Saint Basile G. CD40 ligand mutations in xlinked immunodeficiency with hyper-IgM. Nature 1993;361:541-543. Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J.Biol.Chem. 1998;273:32377-32379. Eeva J, Nuutinen U, Ropponen A, Matto M, Eray M, Pellinen R et al. The involvement of mitochondria and the caspase-9 activation pathway in rituximab-induced apoptosis in FL cells. Apoptosis 2009;14:687-698. Eeva J, Postila V, Matto M, Nuutinen U, Ropponen A, Eray M et al. Kinetics and signaling requirements of CD40-mediated protection from B cell receptor-induced apoptosis. Eur.J.Immunol. 2003;33:2783-2791. Eeva J, Ropponen A, Nuutinen U, Eeva ST, Matto M, Eray M et al. The CD40-induced protection against CD95-mediated apoptosis is associated with a rapid upregulation of anti-apoptotic c-FLIP. Mol.Immunol. 2007;44:1230-1237. Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol.Rev. 2009;229:152-172. Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J.Biol.Chem. 1998;273:14363-14367. Eramo A, Pallini R, Lotti F, Sette G, Patti M, Bartucci M et al. Inhibition of DNA methylation sensitizes glioblastoma for tumor necrosis factor-related apoptosis-inducing ligand-mediated destruction. Cancer Res. 2005;65:11469-11477. Eray M, Postila V, Eeva J, Ripatti A, Karjalainen-Lindsberg ML, Knuutila S et al. Follicular lymphoma cell lines, an in vitro model for antigenic selection and cytokine-mediated growth regulation of germinal centre B cells. Scand.J.Immunol. 2003;57:545-555. Erlacher M, Michalak EM, Kelly PN, Labi V, Niederegger H, Coultas L et al. BH3-only proteins puma and bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo. Blood 2005;106:4131-4138. Fakler M, Loeder S, Vogler M, Schneider K, Jeremias I, Debatin KM et al. Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome bcl-2-mediated resistance. Blood 2009;113:1710-1722.

80 Feinman R, Koury J, Thames M, Barlogie B, Epstein J, Siegel DS. Role of NF-kappaB in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by bcl-2. Blood 1999;93:3044-3052. Franke TF. PI3K/Akt: Getting it right matters. Oncogene 2008;27:6473-6488. Frankfurt O, Rosen ST. Mechanisms of glucocorticoid-induced apoptosis in hematologic malignancies: Updates. Curr.Opin.Oncol. 2004;16:553-563. Fulda S. Inhibitor of apoptosis proteins in hematological malignancies. Leukemia 2009;23:467476. Fulda S, Meyer E, Debatin KM. Inhibition of TRAIL-induced apoptosis by bcl-2 overexpression. Oncogene 2002;21:2283-2294. Funakoshi S, Longo DL, Beckwith M, Conley DK, Tsarfaty G, Tsarfaty I et al. Inhibition of human B-cell lymphoma growth by CD40 stimulation. Blood 1994;83:2787-2794. Galliher-Beckley AJ, Williams JG, Collins JB, Cidlowski JA. Glycogen synthase kinase 3betamediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles. Mol.Cell.Biol. 2008;28:7309-7322. Gaynon PS, Carrel AL. Glucocorticosteroid therapy in childhood acute lymphoblastic leukemia. Adv.Exp.Med.Biol. 1999;457:593-605. Ghia P, Boussiotis VA, Schultze JL, Cardoso AA, Dorfman DM, Gribben JG et al. Unbalanced expression of bcl-2 family proteins in follicular lymphoma: Contribution of CD40 signaling in promoting survival. Blood 1998;91:244-251. Glas AM, Kersten MJ, Delahaye LJ, Witteveen AT, Kibbelaar RE, Velds A et al. Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood 2005;105:301-307. Glick RD, Medary I, Aronson DC, Scotto KW, Swendeman SL, La Quaglia MP. The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines. J.Pediatr.Surg. 2000;35:465-472. Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 2001;15:1406-1418. Goya L, Maiyar AC, Ge Y, Firestone GL. Glucocorticoids induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that is synchronously reversed by steroid withdrawal or addition of transforming growth factor-alpha. Mol.Endocrinol. 1993;7:1121-1132. Green DR. Apoptotic pathways: ten minutes to dead. Cell 2005;121:671-674. Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626629.

81 Greenstein S, Ghias K, Krett NL, Rosen ST. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clin.Cancer Res. 2002;8:1681-1694. Griffith TS, Rauch CT, Smolak PJ, Waugh JY, Boiani N, Lynch DH et al. Functional analysis of TRAIL receptors using monoclonal antibodies. J.Immunol. 1999;162:2597-2605. Grillot DA, Merino R, Nunez G. Bcl-XL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice. J.Exp.Med. 1995;182:1973-1983. Grimes CA, Jope RS. CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium. J.Neurochem. 2001;78:1219-1232. Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998;17:3878-3885. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999;13:1899-1911. Guicciardi ME, Bronk SF, Werneburg NW, Gores GJ. cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis. Am.J.Physiol.Gastrointest.Liver Physiol. 2007;292:G1337-46. Guicciardi ME, Gores GJ. Life and death by death receptors. FASEB J. 2009;23:1625-1637. Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M, Soengas MS et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 1998;94:339-352. Han J, Flemington C, Houghton AB, Gu Z, Zambetti GP, Lutz RJ et al. Expression of bbc3, a proapoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc.Natl.Acad.Sci.U.S.A. 2001;98:11318-11323. Harris CA, Johnson EM,Jr. BH3-only bcl-2 family members are coordinately regulated by the JNK pathway and require bax to induce apoptosis in neurons. J.Biol.Chem. 2001;276:37754-37760. Hartmann BL, Geley S, Loffler M, Hattmannstorfer R, Strasser-Wozak EM, Auer B et al. Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis. Oncogene 1999;18:713-719. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev. 2004;18:2195-2224. Herr I, Gassler N, Friess H, Buchler MW. Regulation of differential pro- and anti-apoptotic signaling by glucocorticoids. Apoptosis 2007;12:271-291. Hinz S, Trauzold A, Boenicke L, Sandberg C, Beckmann S, Bayer E et al. Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene 2000;19:5477-5486. Homig-Holzel C, Hojer C, Rastelli J, Casola S, Strobl LJ, Muller W et al. Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis. J.Exp.Med. 2008;205:1317-1329.

82 Hongisto V, Smeds N, Brecht S, Herdegen T, Courtney MJ, Coffey ET. Lithium blocks the c-jun stress response and protects neurons via its action on glycogen synthase kinase 3. Mol.Cell.Biol. 2003;23:6027-6036. Hopkins-Donaldson S, Bodmer JL, Bourloud KB, Brognara CB, Tschopp J, Gross N. Loss of caspase-8 expression in neuroblastoma is related to malignancy and resistance to TRAIL-induced apoptosis. Med.Pediatr.Oncol. 2000;35:608-611. Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of bax and bcl-X(L) during apoptosis. Proc.Natl.Acad.Sci.U.S.A. 1997;94:3668-3672. Hu L, Hofmann J, Lu Y, Mills GB, Jaffe RB. Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Cancer Res. 2002;62:10871092. Huang DC, Strasser A. BH3-only proteins-essential initiators of apoptotic cell death. Cell 2000;103:839-842. Hunter AM, LaCasse EC, Korneluk RG. The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis 2007;12:1543-1568. Hymowitz SG, O'Connell MP, Ultsch MH, Hurst A, Totpal K, Ashkenazi A et al. A unique zincbinding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 2000;39:633-640. Imaizumi K, Morihara T, Mori Y, Katayama T, Tsuda M, Furuyama T et al. The cell deathpromoting gene DP5, which interacts with the BCL2 family, is induced during neuronal apoptosis following exposure to amyloid beta protein. J.Biol.Chem. 1999;274:7975-7981. Imaizumi K, Tsuda M, Imai Y, Wanaka A, Takagi T, Tohyama M. Molecular cloning of a novel polypeptide, DP5, induced during programmed neuronal death. J.Biol.Chem. 1997;272:1884218848. Inohara N, Ding L, Chen S, Nunez G. Harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins bcl-2 and bclX(L). EMBO J. 1997;16:1686-1694. Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer.Biol.Ther. 2005;4:139-163. Jin ZH, Kurosu T, Yamaguchi M, Arai A, Miura O. Hematopoietic cytokines enhance Chk1dependent G2/M checkpoint activation by etoposide through the Akt/GSK3 pathway to inhibit apoptosis. Oncogene 2005;24:1973-1981. Johnstone RW, Frew AJ, Smyth MJ. The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nat.Rev.Cancer. 2008;8:782-798. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW et al. Glycogen synthase kinase3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J.Clin.Invest. 2004;113:1535-1549.

83 Kane LP, Shapiro VS, Stokoe D, Weiss A. Induction of NF-kappaB by the Akt/PKB kinase. Curr.Biol. 1999;9:601-604. Kehry MR. CD40-mediated signaling in B cells. balancing cell survival, growth, and death. J.Immunol. 1996;156:2345-2348. Kelley RF, Totpal K, Lindstrom SH, Mathieu M, Billeci K, Deforge L et al. Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling. J.Biol.Chem. 2005;280:2205-2212. Kennedy SG, Kandel ES, Cross TK, Hay N. Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol.Cell.Biol. 1999;19:5800-5810. Keogh SA, Walczak H, Bouchier-Hayes L, Martin SJ. Failure of bcl-2 to block cytochrome c redistribution during TRAIL-induced apoptosis. FEBS Lett. 2000;471:93-98. Kim EJ, Suliman A, Lam A, Srivastava RK. Failure of bcl-2 to block mitochondrial dysfunction during TRAIL-induced apoptosis. tumor necrosis-related apoptosis-inducing ligand. Int.J.Oncol. 2001;18:187-194. Kim H, Rafiuddin-Shah M, Tu HC, Jeffers JR, Zambetti GP, Hsieh JJ et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat.Cell Biol. 2006;8:1348-1358. King KL, Cidlowski JA. Cell cycle regulation and apoptosis. Annu.Rev.Physiol. 1998;60:601-617. King TD, Bijur GN, Jope RS. Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res. 2001;919:106-114. Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH et al. Cytotoxicitydependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995;14:5579-5588. Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A. Apo2L/TRAILdependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 2000;12:611-620. Kischkel FC, Lawrence DA, Tinel A, LeBlanc H, Virmani A, Schow P et al. Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J.Biol.Chem. 2001;276:46639-46646. Klein U, Dalla-Favera R. Germinal centres: Role in B-cell physiology and malignancy. Nat.Rev.Immunol. 2008;8:22-33. Knight MJ, Riffkin CD, Ekert PG, Ashley DM, Hawkins CJ. Caspase-8 levels affect necessity for mitochondrial amplification in death ligand-induced glioma cell apoptosis. Mol.Carcinog. 2004;39:173-182. Kofler R, Schmidt S, Kofler A, Ausserlechner MJ. Resistance to glucocorticoid-induced apoptosis in lymphoblastic leukemia. J.Endocrinol. 2003;178:19-27.

84 Korsmeyer SJ. BCL-2 gene family and the regulation of programmed cell death. Cancer Res. 1999;59:1693s-1700s. Korthauer U, Graf D, Mages HW, Briere F, Padayachee M, Malcolm S et al. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature 1993;361:539541. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat.Rev.Cancer. 2005;5:886-897. Krueger A, Schmitz I, Baumann S, Krammer PH, Kirchhoff S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. J.Biol.Chem. 2001;276:20633-20640. Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998;94:325-337. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR et al. BH3 domains of BH3-only proteins differentially regulate bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol.Cell 2005;17:525-535. Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr.Opin.Cell Biol. 2003;15:691-699. Lakhani SA, Masud A, Kuida K, Porter GA,Jr, Booth CJ, Mehal WZ et al. Caspases 3 and 7: Key mediators of mitochondrial events of apoptosis. Science 2006;311:847-851. Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G. NF-kappaB-mediated up-regulation of bcl-x and bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc.Natl.Acad.Sci.U.S.A. 1999;96:9136-9141. Lemaire C, Andreau K, Souvannavong V, Adam A. Specific dual effect of cycloheximide on B lymphocyte apoptosis: Involvement of CPP32/caspase-3. Biochem.Pharmacol. 1999;58:85-93. Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer.Cell. 2002;2:183-192. Leung KT, Li KK, Sun SS, Chan PK, Ooi VE, Chiu LC. Activation of the JNK pathway promotes phosphorylation and degradation of BimEL--a novel mechanism of chemoresistance in T-cell acute lymphoblastic leukemia. Carcinogenesis 2008;29:544-551. Leverkus M, Sprick MR, Wachter T, Mengling T, Baumann B, Serfling E et al. Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by removing the resistancemediating block of effector caspase maturation. Mol.Cell.Biol. 2003;23:777-790. Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the fas pathway of apoptosis. Cell 1998;94:491-501. Li J, Yuan J. Caspases in apoptosis and beyond. Oncogene 2008;27:6194-6206.

85 Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 2001;412:95-99. Liao X, Zhang L, Thrasher JB, Du J, Li B. Glycogen synthase kinase-3beta suppression eliminates tumor necrosis factor-related apoptosis-inducing ligand resistance in prostate cancer. Mol.Cancer.Ther. 2003;2:1215-1222. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA et al. Glycogen synthase kinase-3beta phosphorylates bax and promotes its mitochondrial localization during neuronal apoptosis. J.Neurosci. 2004;24:9993-10002. Liu H, Perlman H, Pagliari LJ, Pope RM. Constitutively activated akt-1 is vital for the survival of human monocyte-differentiated macrophages. role of mcl-1, independent of nuclear factor (NF)kappaB, bad, or caspase activation. J.Exp.Med. 2001;194:113-126. Liu JL, Chiles TC, Sen RJ, Rothstein TL. Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface ig receptor. J.Immunol. 1991;146:1685-1691. Lub-de Hooge MN, de Vries EG, de Jong S, Bijl M. Soluble TRAIL concentrations are raised in patients with systemic lupus erythematosus. Ann.Rheum.Dis. 2005;64:854-858. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 1998;94:481-490. Luthi AU, Martin SJ. The CASBAH: A searchable database of caspase substrates. Cell Death Differ. 2007;14:641-650. MacLennan IC. Germinal centers. Annu.Rev.Immunol. 1994;12:117-139. Majewski N, Nogueira V, Robey RB, Hay N. Akt inhibits apoptosis downstream of BID cleavage via a glucose-dependent mechanism involving mitochondrial hexokinases. Mol.Cell.Biol. 2004;24:730-740. Marani M, Tenev T, Hancock D, Downward J, Lemoine NR. Identification of novel isoforms of the BH3 domain protein bim which directly activate bax to trigger apoptosis. Mol.Cell.Biol. 2002;22:3577-3589. Marchetti MC, Di Marco B, Cifone G, Migliorati G, Riccardi C. Dexamethasone-induced apoptosis of thymocytes: Role of glucocorticoid receptor-associated src kinase and caspase-8 activation. Blood 2003;101:585-593. Marchetti P, Castedo M, Susin SA, Zamzami N, Hirsch T, Macho A et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J.Exp.Med. 1996;184:11551160. Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D et al. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr.Biol. 1997;7:1003-1006.

86 Martelli AM, Tazzari PL, Tabellini G, Bortul R, Billi AM, Manzoli L et al. A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells. Leukemia 2003;17:1794-1805. Martinez A, Carreras J, Campo E. The follicular lymphoma microenvironment: from tumor cell to host immunity. Current Hematologic Malignancy Reports 2008;3:179-186. Matto M, Nuutinen UM, Ropponen A, Myllykangas K, Pelkonen J. CD45RA and RO isoforms have distinct effects on cytokine- and B-cell-receptor-mediated signalling in human B cells. Scand.J.Immunol. 2005;61:520-528. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol.Cell 2006;21:749-760. Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc.Natl.Acad.Sci.U.S.A. 2001;98:11598-11603. Merino D, Lalaoui N, Morizot A, Schneider P, Solary E, Micheau O. Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2. Mol.Cell.Biol. 2006;26:70467055. Micheau O, Thome M, Schneider P, Holler N, Tschopp J, Nicholson DW et al. The long form of FLIP is an activator of caspase-8 at the fas death-inducing signaling complex. J.Biol.Chem. 2002;277:45162-45171. Mitsiades CS, Mitsiades N, Poulaki V, Schlossman R, Akiyama M, Chauhan D et al. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: Therapeutic implications. Oncogene 2002;21:5673-5683. Moalli PA, Rosen ST. Glucocorticoid receptors and resistance to glucocorticoids in hematologic malignancies. Leuk.Lymphoma 1994;15:363-374. Munshi A, Pappas G, Honda T, McDonnell TJ, Younes A, Li Y et al. TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by bcl-2. Oncogene 2001;20:3757-3765. Nachmias B, Ashhab Y, Ben-Yehuda D. The inhibitor of apoptosis protein family (IAPs): An emerging therapeutic target in cancer. Semin.Cancer Biol. 2004;14:231-243. Nagaraj NS, Vigneswaran N, Zacharias W. Cathepsin B mediates TRAIL-induced apoptosis in oral cancer cells. J.Cancer Res.Clin.Oncol. 2006;132:171-183. Nagy K, Szekely-Szuts K, Izeradjene K, Douglas L, Tillman M, Barti-Juhasz H et al. Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria. Pathol.Oncol.Res. 2006;12:133-142. Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol.Cell 2001;7:683-694. Nechushtan A, Smith CL, Hsu YT, Youle RJ. Conformation of the bax C-terminus regulates subcellular location and cell death. EMBO J. 1999;18:2330-2341.

87 Ng SSW, Tsao MS, Chow S, Hedley DW. Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer Res. 2000;60:5451-5455. Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell.Signal. 2002;14:381-395. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T et al. Noxa, a BH3-only member of the bcl-2 family and candidate mediator of p53-induced apoptosis. Science 2000;288:1053-1058. O'Gorman DM, McKenna SL, McGahon AJ, Knox KA, Cotter TG. Sensitisation of HL60 human leukaemic cells to cytotoxic drug-induced apoptosis by inhibition of PI3-kinase survival signals. Leukemia 2000;14:602-611. Oh KJ, Barbuto S, Pitter K, Morash J, Walensky LD, Korsmeyer SJ. A membrane-targeted BID BCL-2 homology 3 peptide is sufficient for high potency activation of BAX in vitro. J.Biol.Chem. 2006;281:36999-37008. Osaki M, Oshimura M, Ito H. PI3K-akt pathway: Its functions and alterations in human cancer. Apoptosis 2004;9:667-676. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. NF-kappaB activation by tumour necrosis factor requires the akt serine-threonine kinase. Nature 1999;401:82-85. Ozoren N, El-Deiry WS. Defining characteristics of types I and II apoptotic cells in response to TRAIL. Neoplasia 2002;4:551-557. Ozoren N, Kim K, Burns TF, Dicker DT, Moscioni AD, El-Deiry WS. The caspase 9 inhibitor ZLEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res. 2000;60:6259-6265. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domaincontaining receptor for TRAIL. Science 1997a;277:815-818. Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J et al. The receptor for the cytotoxic ligand TRAIL. Science 1997b;276:111-113. Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. J.Biol.Chem. 1998;273:19929-19932. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits bax-induced cytochrome c release and apoptosis. J.Biol.Chem. 2002;277:7610-7618. Pedersen KB, Vedeckis WV. Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines. Biochemistry 2003;42:10978-10990. Petit PX, Lecoeur H, Zorn E, Dauguet C, Mignotte B, Gougeon ML. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J.Cell Biol. 1995;130:157-167.

88 Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J.Biol.Chem. 1996;271:12687-12690. Planey SL, Abrams MT, Robertson NM, Litwack G. Role of apical caspases and glucocorticoidregulated genes in glucocorticoid-induced apoptosis of pre-B leukemic cells. Cancer Res. 2003;63:172-178. Pugazhenthi S, Nesterova A, Sable C, Heidenreich KA, Boxer LM, Heasley LE et al. Akt/protein kinase B up-regulates bcl-2 expression through cAMP-response element-binding protein. J.Biol.Chem. 2000;275:10761-10766. Putcha GV, Moulder KL, Golden JP, Bouillet P, Adams JA, Strasser A et al. Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis. Neuron 2001;29:615-628. Puthalakath H, Huang DC, O'Reilly LA, King SM, Strasser A. The proapoptotic activity of the bcl2 family member bim is regulated by interaction with the dynein motor complex. Mol.Cell 1999;3:287-296. Puthalakath H, Villunger A, O'Reilly LA, Beaumont JG, Coultas L, Cheney RE et al. Bmf: A proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis. Science 2001;293:1829-1832. Rambal AA, Panaguiton ZL, Kramer L, Grant S, Harada H. MEK inhibitors potentiate dexamethasone lethality in acute lymphoblastic leukemia cells through the pro-apoptotic molecule BIM. Leukemia 2009;. Ramdas J, Liu W, Harmon JM. Glucocorticoid-induced cell death requires autoinduction of glucocorticoid receptor expression in human leukemic T cells. Cancer Res. 1999;59:1378-1385. Renner K, Ausserlechner MJ, Kofler R. A conceptual view on glucocorticoid-lnduced apoptosis, cell cycle arrest and glucocorticoid resistance in lymphoblastic leukemia. Curr.Mol.Med. 2003;3:707-717. Ricci MS, Kim SH, Ogi K, Plastaras JP, Ling J, Wang W et al. Reduction of TRAIL-induced mcl-1 and cIAP2 by c-myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer.Cell. 2007;12:66-80. Riedl SJ, Salvesen GS. The apoptosome: Signalling platform of cell death. Nat.Rev.Mol.Cell Biol. 2007;8:405-413. Riml S, Schmidt S, Ausserlechner MJ, Geley S, Kofler R. Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in jurkat acute lymphoblastic leukemia cells. Cell Death Differ. 2004;11 Suppl 1:S65-72. Rokhlin OW, Guseva N, Tagiyev A, Knudson CM, Cohen MB. Bcl-2 oncoprotein protects the human prostatic carcinoma cell line PC3 from TRAIL-mediated apoptosis. Oncogene 2001;20:2836-2843.

89 Rosato RR, Almenara JA, Coe S, Grant S. The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with mcl-1 and cFLIPL down-regulation. Cancer Res. 2007;67:9490-9500. Rottmann S, Wang Y, Nasoff M, Deveraux QL, Quon KC. A TRAIL receptor-dependent synthetic lethal relationship between MYC activation and GSK3beta/FBW7 loss of function. Proc.Natl.Acad.Sci.U.S.A. 2005;102:15195-15200. Roue G, Perez-Galan P, Lopez-Guerra M, Villamor N, Campo E, Colomer D. Selective inhibition of IkappaB kinase sensitizes mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level. J.Immunol. 2007;178:1923-1930. Rudner J, Jendrossek V, Lauber K, Daniel PT, Wesselborg S, Belka C. Type I and type II reactions in TRAIL-induced apoptosis -- results from dose-response studies. Oncogene 2005;24:130-140. Rudner J, Lepple-Wienhues A, Budach W, Berschauer J, Friedrich B, Wesselborg S et al. Wildtype, mitochondrial and ER-restricted bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis. J.Cell.Sci. 2001;114:4161-4172. Ruiz de Almodovar C, Ruiz-Ruiz C, Munoz-Pinedo C, Robledo G, Lopez-Rivas A. The differential sensitivity of Bc1-2-overexpressing human breast tumor cells to TRAIL or doxorubicin-induced apoptosis is dependent on Bc1-2 protein levels. Oncogene 2001;20:7128-7133. Samraj AK, Keil E, Ueffing N, Schulze-Osthoff K, Schmitz I. Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis. J.Biol.Chem. 2006;281:2965229659. Samuel T, Welsh K, Lober T, Togo SH, Zapata JM, Reed JC. Distinct BIR domains of cIAP1 mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and second mitochondrial activator of caspases. J.Biol.Chem. 2006;281:1080-1090. Sanchez I, Goya L, Vallerga AK, Firestone GL. Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression. Cell Growth Differ. 1993;4:215225. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998;17:1675-1687. Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J.Biol.Chem. 1999;274:1541-1548. Schmaltz C, Alpdogan O, Kappel BJ, Muriglan SJ, Rotolo JA, Ongchin J et al. T cells require TRAIL for optimal graft-versus-tumor activity. Nat.Med. 2002;8:1433-1437. Schmidt S, Rainer J, Ploner C, Presul E, Riml S, Kofler R. Glucocorticoid-induced apoptosis and glucocorticoid resistance: Molecular mechanisms and clinical relevance. Cell Death Differ. 2004;11 Suppl 1:S45-55.

90 Schwabe RF, Brenner DA. Role of glycogen synthase kinase-3 in TNF-alpha-induced NF-kappaB activation and apoptosis in hepatocytes. Am.J.Physiol.Gastrointest.Liver Physiol. 2002;283:G20411. Seki N, Hayakawa Y, Brooks AD, Wine J, Wiltrout RH, Yagita H et al. Tumor necrosis factorrelated apoptosis-inducing ligand-mediated apoptosis is an important endogenous mechanism for resistance to liver metastases in murine renal cancer. Cancer Res. 2003;63:207-213. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D et al. Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 1997;277:818-821. Shinjyo T, Kuribara R, Inukai T, Hosoi H, Kinoshita T, Miyajima A et al. Downregulation of bim, a proapoptotic relative of bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors. Mol.Cell.Biol. 2001;21:854-864. Skommer J, Wlodkowic D, Deptala A. Larger than life: Mitochondria and the bcl-2 family. Leuk.Res. 2007;31:277-286. Skommer J, Wlodkowic D, Matto M, Eray M, Pelkonen J. HA14-1, a small molecule bcl-2 antagonist, induces apoptosis and modulates action of selected anticancer drugs in follicular lymphoma B cells. Leuk.Res. 2006;30:322-331. Smets LA, Salomons G, van den Berg J. Glucocorticoid induced apoptosis in leukemia. Adv.Exp.Med.Biol. 1999;457:607-614. Song L, Zhou T, Jope RS. Lithium facilitates apoptotic signaling induced by activation of the fas death domain-containing receptor. BMC Neurosci. 2004;5:20. Sprick MR, Rieser E, Stahl H, Grosse-Wilde A, Weigand MA, Walczak H. Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADDdependent manner but can not functionally substitute caspase-8. EMBO J. 2002;21:4520-4530. Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J et al. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 2000;12:599-609. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by apaf-1-mediated oligomerization. Mol.Cell 1998;1:949-957. Srinivasula SM, Datta P, Fan XJ, Fernandes-Alnemri T, Huang Z, Alnemri ES. Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. J.Biol.Chem. 2000;275:36152-36157. Stavnezer J. Molecular processes that regulate class switching. Curr.Top.Microbiol.Immunol. 2000;245:127-168. Strasser A. The role of BH3-only proteins in the immune system. Nat.Rev.Immunol. 2005;5:189200. Strasser A, Harris AW, Cory S. Bcl-2 transgene inhibits T cell death and perturbs thymic selfcensorship. Cell 1991;67:889-899.

91 Strasser A, Harris AW, Huang DC, Krammer PH, Cory S. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J. 1995;14:6136-6147. Suliman A, Lam A, Datta R, Srivastava RK. Intracellular mechanisms of TRAIL: Apoptosis through mitochondrial-dependent and -independent pathways. Oncogene 2001;20:2122-2133. Sun SY, Yue P, Zhou JY, Wang Y, Choi Kim HR, Lotan R et al. Overexpression of BCL2 blocks TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. Biochem.Biophys.Res.Commun. 2001;280:788-797. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441-446. Tajes M, Yeste-Velasco M, Zhu X, Chou SP, Smith MA, Pallàs M et al. Activation of Akt by lithium: pro-survival pathways in aging. Mech Ageing Dev. 2009;130:253-261. Takeda K, Hayakawa Y, Smyth MJ, Kayagaki N, Yamaguchi N, Kakuta S et al. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells. Nat.Med. 2001;7:94-100. Tan J, Geng L, Yazlovitskaya EM, Hallahan DE. Protein kinase B/Akt-dependent phosphorylation of glycogen synthase kinase-3beta in irradiated vascular endothelium. Cancer Res. 2006;66:23202327. Tan J, Zhuang L, Leong HS, Iyer NG, Liu ET, Yu Q. Pharmacologic modulation of glycogen synthase kinase-3beta promotes p53-dependent apoptosis through a direct bax-mediated mitochondrial pathway in colorectal cancer cells. Cancer Res. 2005;65:9012-9020. Travert M, Ame-Thomas P, Pangault C, Morizot A, Micheau O, Semana G et al. CD40 ligand protects from TRAIL-induced apoptosis in follicular lymphomas through NF-kappaB activation and up-regulation of c-FLIP and bcl-xL. J.Immunol. 2008;181:1001-1011. Truneh A, Sharma S, Silverman C, Khandekar S, Reddy MP, Deen KC et al. Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor. J.Biol.Chem. 2000;275:23319-23325. van Delft MF, Huang DC. How the bcl-2 family of proteins interact to regulate apoptosis. Cell Res. 2006;16:203-213. van Kooten C, Banchereau J. CD40-CD40 ligand. J.Leukoc.Biol. 2000;67:2-17. Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 1993;75:229-240. Verma S, Zhao LJ, Chinnadurai G. Phosphorylation of the pro-apoptotic protein BIK: Mapping of phosphorylation sites and effect on apoptosis. J.Biol.Chem. 2001;276:4671-4676. Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M et al. Targeting XIAP bypasses bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo. Cancer Res. 2008;68:7956-7965.

92 Wagner KW, Punnoose EA, Januario T, Lawrence DA, Pitti RM, Lancaster K et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat.Med. 2007;13:1070-1077. Walczak H, Bouchon A, Stahl H, Krammer PH. Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on bcl-2- or bcl-xL-overexpressing chemotherapyresistant tumor cells. Cancer Res. 2000;60:3051-3057. Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N et al. TRAIL-R2: A novel apoptosis-mediating receptor for TRAIL. EMBO J. 1997;16:5386-5397. Walensky LD, Pitter K, Morash J, Oh KJ, Barbuto S, Fisher J et al. A stapled BID BH3 helix directly binds and activates BAX. Mol.Cell 2006;24:199-210. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS,Jr. NF-kappaB antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680-1683. Wang D, Muller N, McPherson KG, Reichardt HM. Glucocorticoids engage different signal transduction pathways to induce apoptosis in thymocytes and mature T cells. J.Immunol. 2006;176:1695-1702. Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, Yang-Yen HF. The antiapoptotic gene mcl-1 is upregulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol.Cell.Biol. 1999;19:6195-6206. Wang Q, Li N, Wang X, Kim MM, Evers BM. Augmentation of sodium butyrate-induced apoptosis by phosphatidylinositol 3'-kinase inhibition in the KM20 human colon cancer cell line. Clin.Cancer Res. 2002;8:1940-1947. Wang S, Wang S, Yang T, Zhu F, Zhu J, Huang Y et al. CD40L-mediated inhibition of NF-kappaB in CA46 burkitt lymphoma cells promotes apoptosis. Leuk.Lymphoma 2008;49:1792-1799. Wang Z, Malone MH, He H, McColl KS, Distelhorst CW. Microarray analysis uncovers the induction of the proapoptotic BH3-only protein bim in multiple models of glucocorticoid-induced apoptosis. J.Biol.Chem. 2003;278:23861-23867. Watcharasit P, Bijur GN, Song L, Zhu J, Chen X, Jope RS. Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53. J.Biol.Chem. 2003;278:48872-48879. Watcharasit P, Bijur GN, Zmijewski JW, Song L, Zmijewska A, Chen X et al. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc.Natl.Acad.Sci.U.S.A. 2002;99:7951-7955. Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR. Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J.Cell Biol. 2001;153:319-328. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 2000;14:2060-2071.

93 Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ et al. Proapoptotic BAX and BAK: A requisite gateway to mitochondrial dysfunction and death. Science 2001;292:727-730. Weiss LM, Warnke RA, Sklar J, Cleary ML. Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas. N.Engl.J.Med. 1987;317:1185-1189. Werneburg NW, Guicciardi ME, Bronk SF, Kaufmann SH, Gores GJ. Tumor necrosis factorrelated apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by bcl-2 proteins. J.Biol.Chem. 2007;282:28960-28970. West KA, Castillo SS, Dennis PA. Activation of the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resist Updat 2002;5:234-248. Whitfield J, Neame SJ, Paquet L, Bernard O, Ham J. Dominant-negative c-jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release. Neuron 2001;29:629-643. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673682. Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI et al. Proapoptotic bak is sequestered by mcl-1 and bcl-xL, but not bcl-2, until displaced by BH3-only proteins. Genes Dev. 2005;19:12941305. Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE et al. Apoptosis initiated when BH3 ligands engage multiple bcl-2 homologs, not bax or bak. Science 2007;315:856-859. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of bax from the cytosol to mitochondria during apoptosis. J.Cell Biol. 1997;139:1281-1292. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood 2003;102:972-980. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B et al. Bid-deficient mice are resistant to fas-induced hepatocellular apoptosis. Nature 1999;400:886-891. Yoshino T, Kishi H, Nagata T, Tsukada K, Saito S, Muraguchi A. Differential involvement of p38 MAP kinase pathway and bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes. Eur.J.Immunol. 2001;31:2702-2708. You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M et al. FOXO3adependent regulation of puma in response to cytokine/growth factor withdrawal. J.Exp.Med. 2006;203:1657-1663. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol.Cell 2001;7:673-682. Yun SI, Yoon HY, Jeong SY, Chung YS. Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3beta. J.Bone Miner.Metab. 2009;27:140-148.

94 Zerafa N, Westwood JA, Cretney E, Mitchell S, Waring P, Iezzi M et al. Cutting edge: TRAIL deficiency accelerates hematological malignancies. J.Immunol. 2005;175:5586-5590. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 1996;87:619-628. Zha J, Weiler S, Oh KJ, Wei MC, Korsmeyer SJ. Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 2000;290:1761-1765. Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS. Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. J.Biol.Chem. 2003;278:33067-33077. Zhang L, Insel PA. The pro-apoptotic protein bim is a convergence point for cAMP/protein kinase A- and glucocorticoid-promoted apoptosis of lymphoid cells. J.Biol.Chem. 2004;279:20858-20865. Zhao Y, Difrancesca D, Wang X, Zarnegar R, Michalopoulos GK, Yin XM. Promotion of fasmediated apoptosis in type II cells by high doses of hepatocyte growth factor bypasses the mitochondrial requirement. J.Cell.Physiol. 2007;213:556-563. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC. HER-2/neu induces p53 ubiquitination via akt-mediated MDM2 phosphorylation. Nat.Cell Biol. 2001;3:973-982. Zhou Y, Uddin S, Zimmerman T, Kang JA, Ulaszek J, Wickrema A. Growth control of multiple myeloma cells through inhibition of glycogen synthase kinase-3. Leuk.Lymphoma 2008;49:19451953. Zhu H, Guo W, Zhang L, Wu S, Teraishi F, Davis JJ et al. Proteasome inhibitors-mediated TRAIL resensitization and bik accumulation. Cancer.Biol.Ther. 2005;4:781-786. Zong WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB. BH3-only proteins that bind prosurvival bcl-2 family members fail to induce apoptosis in the absence of bax and bak. Genes Dev. 2001;15:1481-1486. Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J.Biol.Chem. 1999;274:11549-11556.

Kuopio University Publications D. Medical Sciences D 434. Hassinen, Maija. Predictors and consequences of the metabolic syndrome: population-based studies in aging men and women. 2008. Acad. Diss. D 435. Saltevo, Juha. Low-grade inflammation and adiponectin in the metabolic syndrome. 2008. 109 p. Acad. Diss. D 436. Ervasti, Mari. Evaluation of Iron Status Using Methods Based on the Features of Red Blood Cells and Reticulocytes. 2008. 104 p. Acad. Diss. D 437. Muukka, Eija. Luomun tie päiväkotiin: luomuruokailun toteutettavuus ja ravitsemuksellinen merkitys päiväkotilapsille. 2008. 168 p. Acad. Diss. D 438. Sörensen, Lars. Work ability and health-related quality of life in middle-aged men: the role of physical activity and fitness. 2008. 83 p. Acad. Diss. D 439. Maaranen, Päivi. Dissociation in the finnish general population. 2008. 97 p. Acad. Diss. D 440. Hyvönen, Juha. Suomen psykiatrinen hoitojärjestelmä 1990-luvulla historian jatkumon näkökulmasta. 2008. 279 p. Acad. Diss. D 441. Mäkinen, Heidi. Disease activity and remission in rheumatoid arthritis: comparison of available disease activity measures and development of a novel disease sctivity indes: the mean overall index for rheumatoid arthritis (MOI-RA). 2008. 129 p. Acad. Diss. D 442. Kousa, Anne. The regional association of the hardness in well waters and the incidence of acute myocardial infarction in rural Finland. 2008. 92 p. Acad. Diss. D 443. Olkku, Anu. Glucocorticoid-induced changes in osteoblastic cells: cross-talk with wnt and glutamate signalling pathways. 2009. 118 p. Acad. Diss. D 444. Mattila, Riikka. Effectiveness of a multidisciplinary lifestyle intervention on hypertension, cardiovascular risk factors and musculoskeletal symptoms. 2009. 92 p. Acad. Diss. D 445. Hartmann-Petersen, Susanna. Hyaluronan and CD44 in epidermis with special reference to growth factors and malignant transformation. 2009. 103 p. Acad. Diss. D 446. Tolppanen, Anna-Maija. Genetic association of the tenomodulin gene (TNMD) with obesity- and inflammation-related phenotypes. 2009. 111 p. Acad. Diss. D 447. Lehto, Soili Marianne. Biological findings in major depressive disorder with special reference to the atypical features subtype. 2009. 115 p. Acad. Diss. D 448. Nieminen, Jyrki. Effect of functional loading on remodelling in canine, and normal and collagen type II transgenic murine bone. 2009. 107 p. Acad. Diss. D 449. Torpström, Jaana. Yliopistokoulutus ravitsemusasiantuntijuuden kehittäjänä. 2009. 164 p. Acad. Diss.